The resazurin assay, also known as the Alamar Blue assay, is a widely used technique in cell biology, microbiology, and drug development to assess cell viability through the conversion of resazurin to the fluorescent compound resorufin. This assay plays a crucial role in screening potential drug candidates and evaluating pharmaceutical and chemical toxicity. However, inconsistencies in pharmacogenomic studies have highlighted the need for standardized and optimized laboratory protocols to ensure reliable and consistent results. This manuscript presents a standardized procedure for optimizing the resazurin-based viability assay to enhance the reliability of cytotoxicity data. The optimization focuses on critical experimental parameters such as excitation and emission wavelengths, incubation time, and assay limits, aiming to achieve a measurement imprecision of less than 20%. The approach also emphasizes data quality assessment, including repeatability, reproducibility, and measurement uncertainty. The optimization workflow includes detailed steps for selecting optimal excitation and emission wavelengths, determining the limit of blank, detection, and quantification, and evaluating repeatability and reproducibility. The manuscript provides a comprehensive standard operating procedure (SOP) that covers experimental setup, data analysis, and outlier management. This SOP is designed to improve the robustness and reliability of viability testing results, fostering greater confidence in research findings and advancing the translational potential of preclinical studies. In conclusion, the presented SOP offers a systematic approach to optimize the resazurin assay, addressing challenges of variability and enhancing reliability. By adhering to standardized protocols and optimizing parameters, researchers can ensure consistent and reproducible results, advancing the reliability of viability testing in preclinical drug screening.The resazurin assay, also known as the Alamar Blue assay, is a widely used technique in cell biology, microbiology, and drug development to assess cell viability through the conversion of resazurin to the fluorescent compound resorufin. This assay plays a crucial role in screening potential drug candidates and evaluating pharmaceutical and chemical toxicity. However, inconsistencies in pharmacogenomic studies have highlighted the need for standardized and optimized laboratory protocols to ensure reliable and consistent results. This manuscript presents a standardized procedure for optimizing the resazurin-based viability assay to enhance the reliability of cytotoxicity data. The optimization focuses on critical experimental parameters such as excitation and emission wavelengths, incubation time, and assay limits, aiming to achieve a measurement imprecision of less than 20%. The approach also emphasizes data quality assessment, including repeatability, reproducibility, and measurement uncertainty. The optimization workflow includes detailed steps for selecting optimal excitation and emission wavelengths, determining the limit of blank, detection, and quantification, and evaluating repeatability and reproducibility. The manuscript provides a comprehensive standard operating procedure (SOP) that covers experimental setup, data analysis, and outlier management. This SOP is designed to improve the robustness and reliability of viability testing results, fostering greater confidence in research findings and advancing the translational potential of preclinical studies. In conclusion, the presented SOP offers a systematic approach to optimize the resazurin assay, addressing challenges of variability and enhancing reliability. By adhering to standardized protocols and optimizing parameters, researchers can ensure consistent and reproducible results, advancing the reliability of viability testing in preclinical drug screening.