This review presents a standardized operating procedure (SOP) to optimize resazurin-based viability assays for improved reliability in cytotoxicity studies. Resazurin, also known as Alamar Blue, is a non-toxic blue dye used to assess cell viability by measuring the conversion of resazurin to resorufin, a highly fluorescent compound. The fluorescence intensity (FI) is directly proportional to the number of viable cells. The resazurin assay is more sensitive and has a wider dynamic range compared to tetrazolium assays, making it a valuable tool in drug development and preclinical research. The study highlights the importance of standardizing laboratory protocols to ensure consistent and accurate results in cytotoxicity studies. Variability in results can arise from factors such as cell-specific dehydrogenase and reductase activity, excitation and emission wavelengths, incubation time, and cell concentration. To address these issues, the manuscript outlines an SOP that focuses on optimizing critical parameters, including the selection of optimal excitation and emission wavelengths, incubation time, and the definition of assay limits. The SOP also includes procedures for evaluating repeatability, reproducibility, and measurement uncertainty, ensuring the reliability of the results. The optimization workflow includes steps for determining the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ) using a calibration curve. These parameters are essential for ensuring the accuracy and precision of the assay. The SOP also provides guidelines for outlier management and statistical significance assessment, ensuring the robustness of the results. The study emphasizes the importance of standardized protocols in cell biology to ensure consistency, accuracy, and reproducibility in experimental results. By following the SOP, researchers can enhance the reliability of viability testing, contributing to the advancement of drug safety assessments and the translation of research findings into clinical applications. The manuscript provides a comprehensive approach to optimizing resazurin-based viability assays, ensuring the credibility of cytotoxicity studies and promoting the development of effective therapeutic interventions.This review presents a standardized operating procedure (SOP) to optimize resazurin-based viability assays for improved reliability in cytotoxicity studies. Resazurin, also known as Alamar Blue, is a non-toxic blue dye used to assess cell viability by measuring the conversion of resazurin to resorufin, a highly fluorescent compound. The fluorescence intensity (FI) is directly proportional to the number of viable cells. The resazurin assay is more sensitive and has a wider dynamic range compared to tetrazolium assays, making it a valuable tool in drug development and preclinical research. The study highlights the importance of standardizing laboratory protocols to ensure consistent and accurate results in cytotoxicity studies. Variability in results can arise from factors such as cell-specific dehydrogenase and reductase activity, excitation and emission wavelengths, incubation time, and cell concentration. To address these issues, the manuscript outlines an SOP that focuses on optimizing critical parameters, including the selection of optimal excitation and emission wavelengths, incubation time, and the definition of assay limits. The SOP also includes procedures for evaluating repeatability, reproducibility, and measurement uncertainty, ensuring the reliability of the results. The optimization workflow includes steps for determining the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ) using a calibration curve. These parameters are essential for ensuring the accuracy and precision of the assay. The SOP also provides guidelines for outlier management and statistical significance assessment, ensuring the robustness of the results. The study emphasizes the importance of standardized protocols in cell biology to ensure consistency, accuracy, and reproducibility in experimental results. By following the SOP, researchers can enhance the reliability of viability testing, contributing to the advancement of drug safety assessments and the translation of research findings into clinical applications. The manuscript provides a comprehensive approach to optimizing resazurin-based viability assays, ensuring the credibility of cytotoxicity studies and promoting the development of effective therapeutic interventions.