This study presents a rational approach to optimize structure-switching aptamers (SSAs) for biosensing applications, focusing on cortisol binding. The research aims to improve aptamer binding affinity and introduce target-dependent conformation-switching for real-world biosensor use. Key structural features were identified using NMR and computational modeling to optimize conformational switching in the presence of the target. Large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure seamless transition to a biosensing platform. The study demonstrates initial proof-of-concept with a cortisol binding aptamer, which can be translated to other relevant aptamers. Cortisol, a hormone linked to stress response, is present at quantifiable levels in accessible biofluids. Continuous cortisol monitoring enables real-time tracking of stress levels, crucial for assessing human health and performance. The design of rapid, continuous, reagentless, and noninvasive sensors remains a challenge due to the high sensitivity and specificity required for sensing in biofluids. Structure-switching aptamers (SSAs) can undergo conformational changes in the presence of their target molecule, enabling signal transduction for biosensor platforms. However, converting traditional aptamers into SSAs remains challenging and relies on trial and error. Recent advances include a high-throughput screening platform for converting traditional aptamers into SSAs. The study introduces a robust and rational aptamer optimization strategy that introduces reporter-independent conformation-switching capability and improves target affinity in biofluids relevant to continuous sensing applications. Structural characterization using NMR and computer modeling identified sequence-specific binding features and conformations. NMR also visualizes aptamer conformational changes in the presence and absence of a target, streamlining optimization of switching behavior for sensing applications. A microarray-based platform was used for large-scale mutational analysis, allowing simultaneous assessment of all possible aptamer point mutations and rapid screening of combinatorial mutations for improved binding affinity. Bio-Layer Interferometry (BLI) was used to determine equilibrium dissociation constants for each aptamer to target molecule. All work was performed in a biologically relevant buffer, and the optimized sequence performed well in human biofluid. The study focused on cortisol as a model system, highlighting the importance of continuous, real-time monitoring of stress hormones for preventing negative performance outcomes and diagnosing disease. Cortisol plays a role in the stress-responsive hypothalamus-pituitary-adrenal (HPA) axis and is involved in various bodily processes. Cortisol dysregulation is linked to several conditions, including fatigue, obesity, and depression. Cortisol is present at nanomolar concentrations in blood and has been correlated with other bodily fluids. The study's findings suggest that advances in ssDNA modeling will enable more robust sequence optimization efforts in the future. The approach can be broadly applied to optimize SSThis study presents a rational approach to optimize structure-switching aptamers (SSAs) for biosensing applications, focusing on cortisol binding. The research aims to improve aptamer binding affinity and introduce target-dependent conformation-switching for real-world biosensor use. Key structural features were identified using NMR and computational modeling to optimize conformational switching in the presence of the target. Large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure seamless transition to a biosensing platform. The study demonstrates initial proof-of-concept with a cortisol binding aptamer, which can be translated to other relevant aptamers. Cortisol, a hormone linked to stress response, is present at quantifiable levels in accessible biofluids. Continuous cortisol monitoring enables real-time tracking of stress levels, crucial for assessing human health and performance. The design of rapid, continuous, reagentless, and noninvasive sensors remains a challenge due to the high sensitivity and specificity required for sensing in biofluids. Structure-switching aptamers (SSAs) can undergo conformational changes in the presence of their target molecule, enabling signal transduction for biosensor platforms. However, converting traditional aptamers into SSAs remains challenging and relies on trial and error. Recent advances include a high-throughput screening platform for converting traditional aptamers into SSAs. The study introduces a robust and rational aptamer optimization strategy that introduces reporter-independent conformation-switching capability and improves target affinity in biofluids relevant to continuous sensing applications. Structural characterization using NMR and computer modeling identified sequence-specific binding features and conformations. NMR also visualizes aptamer conformational changes in the presence and absence of a target, streamlining optimization of switching behavior for sensing applications. A microarray-based platform was used for large-scale mutational analysis, allowing simultaneous assessment of all possible aptamer point mutations and rapid screening of combinatorial mutations for improved binding affinity. Bio-Layer Interferometry (BLI) was used to determine equilibrium dissociation constants for each aptamer to target molecule. All work was performed in a biologically relevant buffer, and the optimized sequence performed well in human biofluid. The study focused on cortisol as a model system, highlighting the importance of continuous, real-time monitoring of stress hormones for preventing negative performance outcomes and diagnosing disease. Cortisol plays a role in the stress-responsive hypothalamus-pituitary-adrenal (HPA) axis and is involved in various bodily processes. Cortisol dysregulation is linked to several conditions, including fatigue, obesity, and depression. Cortisol is present at nanomolar concentrations in blood and has been correlated with other bodily fluids. The study's findings suggest that advances in ssDNA modeling will enable more robust sequence optimization efforts in the future. The approach can be broadly applied to optimize SS