This review discusses fluorescence-based methods for monitoring protein stability, focusing on thermal shift assays (TSAs) and isothermal chemical denaturation (ICD). Proteins must maintain their correct structure to function properly, and their stability is influenced by environmental factors. Label-free methods, such as TSAs and ICD, are preferred for their simplicity and low instrument requirements. TSAs, also known as differential scanning fluorimetry (DSF) or ThermoFluor, use fluorescent dyes to monitor protein unfolding and stability. DSF is cost-effective, fast, and suitable for high-throughput screening, making it widely used in research and drug development. It measures changes in fluorescence as proteins unfold, providing insights into stability and interactions. ICD, on the other hand, uses chemical denaturants to study protein stability. Both methods have advantages and limitations, with DSF being more accessible and widely used. The review also discusses other methods like circular dichroism (CD), nuclear magnetic resonance (NMR), and differential scanning calorimetry (DSC), which are more equipment-intensive but provide detailed thermodynamic data. Fluorescent dyes such as SYPRO Orange and ThT are commonly used in TSAs, while intrinsic fluorescence from amino acids like tryptophan can also be used. The review highlights the importance of buffer conditions, as they significantly affect protein stability and assay results. Proper optimization of buffers and dyes is essential for accurate measurements. The review concludes that fluorescence-based methods, particularly DSF, are valuable tools for studying protein stability, with ongoing developments aimed at improving sensitivity and applicability.This review discusses fluorescence-based methods for monitoring protein stability, focusing on thermal shift assays (TSAs) and isothermal chemical denaturation (ICD). Proteins must maintain their correct structure to function properly, and their stability is influenced by environmental factors. Label-free methods, such as TSAs and ICD, are preferred for their simplicity and low instrument requirements. TSAs, also known as differential scanning fluorimetry (DSF) or ThermoFluor, use fluorescent dyes to monitor protein unfolding and stability. DSF is cost-effective, fast, and suitable for high-throughput screening, making it widely used in research and drug development. It measures changes in fluorescence as proteins unfold, providing insights into stability and interactions. ICD, on the other hand, uses chemical denaturants to study protein stability. Both methods have advantages and limitations, with DSF being more accessible and widely used. The review also discusses other methods like circular dichroism (CD), nuclear magnetic resonance (NMR), and differential scanning calorimetry (DSC), which are more equipment-intensive but provide detailed thermodynamic data. Fluorescent dyes such as SYPRO Orange and ThT are commonly used in TSAs, while intrinsic fluorescence from amino acids like tryptophan can also be used. The review highlights the importance of buffer conditions, as they significantly affect protein stability and assay results. Proper optimization of buffers and dyes is essential for accurate measurements. The review concludes that fluorescence-based methods, particularly DSF, are valuable tools for studying protein stability, with ongoing developments aimed at improving sensitivity and applicability.