This report summarizes the findings of two events: a 1990 conference on bioanalytical method validation and a 2000 workshop on the same topic, updated with a decade of progress. The 2000 workshop focused on small molecules, while a separate workshop in March 2000 addressed macromolecules. The purpose of the report is to summarize the progress in analytical methodologies over the past decade, assess key agreements and issues related to small molecules, and provide guiding principles for validating bioanalytical methods used in bioavailability, bioequivalence, and pharmacokinetic studies. The objectives of the conference and workshop were to agree on validation requirements, determine application processes, review progress in bioanalytical methods, identify scientific issues, evaluate comments on the FDA draft guidance, and develop an updated report on bioanalytical method validation. The report discusses acceptable practices for documenting and validating bioanalytical methods, including hyphenated mass spectrometric-based assays, ligand-based assays, high-throughput systems, and the measurement and stability of drug metabolites. It emphasizes the importance of stereoselective determinations and the need for clearly defined validation criteria for each analyte in different biological matrices. The report highlights advancements in mass spectrometry, particularly the widespread use of hyphenated mass spectrometry (LC-MS-MS), which has largely replaced conventional HPLC, GC, and GC-MS assays. It also notes the increasing use of multi-well plates, automated sample processing, and electronic data reporting. Despite these technological advances, the need for clear validation criteria for each analyte in each biological matrix remains. The report defines three types of validation: full validation, partial validation, and cross validation, each with specific requirements depending on the context. Full validation is necessary for new methods or drug entities, while partial validation is used for modifications that do not require full re-validation. Cross validation is used when comparing methods across different laboratories.This report summarizes the findings of two events: a 1990 conference on bioanalytical method validation and a 2000 workshop on the same topic, updated with a decade of progress. The 2000 workshop focused on small molecules, while a separate workshop in March 2000 addressed macromolecules. The purpose of the report is to summarize the progress in analytical methodologies over the past decade, assess key agreements and issues related to small molecules, and provide guiding principles for validating bioanalytical methods used in bioavailability, bioequivalence, and pharmacokinetic studies. The objectives of the conference and workshop were to agree on validation requirements, determine application processes, review progress in bioanalytical methods, identify scientific issues, evaluate comments on the FDA draft guidance, and develop an updated report on bioanalytical method validation. The report discusses acceptable practices for documenting and validating bioanalytical methods, including hyphenated mass spectrometric-based assays, ligand-based assays, high-throughput systems, and the measurement and stability of drug metabolites. It emphasizes the importance of stereoselective determinations and the need for clearly defined validation criteria for each analyte in different biological matrices. The report highlights advancements in mass spectrometry, particularly the widespread use of hyphenated mass spectrometry (LC-MS-MS), which has largely replaced conventional HPLC, GC, and GC-MS assays. It also notes the increasing use of multi-well plates, automated sample processing, and electronic data reporting. Despite these technological advances, the need for clear validation criteria for each analyte in each biological matrix remains. The report defines three types of validation: full validation, partial validation, and cross validation, each with specific requirements depending on the context. Full validation is necessary for new methods or drug entities, while partial validation is used for modifications that do not require full re-validation. Cross validation is used when comparing methods across different laboratories.