This report synthesizes the proceedings of two significant events: the 1990 conference on Analytical Methods Validation—Bioavailability, Bioequivalence and Pharmacokinetic Studies, and the 2000 workshop on "Bioanalytical Methods Validation—A Revisit with a Decade of Progress." The workshop, sponsored by the American Association of Pharmaceutical Scientists and the U.S. Food and Drug Administration, focused on small molecules, while a separate workshop in March 2000 discussed validation principles for macromolecules. The report aims to highlight the progress in analytical methodologies over the past decade 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 the requirements for bioanalytical method validation and to establish procedures for validation. Key topics included acceptable practices for documenting and validating methods, the importance of hyphenated mass spectrometric-based assays, ligand-based assays, high-throughput systems, and the measurement and stability of drug metabolites. The need for stereoselective determinations was also discussed. The introduction emphasizes the critical role of well-characterized and fully validated analytical methods in generating reliable data for bioavailability, bioequivalence, and pharmacokinetic studies. It highlights the ongoing advancements in technology and the need for clearly defined validation criteria, especially for hyphenated mass spectrometry techniques like LC-MS-MS. Bioanalytical method validation involves demonstrating the reliability of a method for quantifying analytes in biological matrices. The report outlines different levels of validation, including Full Validation, Partial Validation, and Cross Validation. Full Validation is necessary for the initial development and implementation of a method, for new drug entities, and for any modifications that add new analytes. Partial Validation covers modifications such as method transfers between laboratories, instrument changes, and changes in matrix or species.This report synthesizes the proceedings of two significant events: the 1990 conference on Analytical Methods Validation—Bioavailability, Bioequivalence and Pharmacokinetic Studies, and the 2000 workshop on "Bioanalytical Methods Validation—A Revisit with a Decade of Progress." The workshop, sponsored by the American Association of Pharmaceutical Scientists and the U.S. Food and Drug Administration, focused on small molecules, while a separate workshop in March 2000 discussed validation principles for macromolecules. The report aims to highlight the progress in analytical methodologies over the past decade 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 the requirements for bioanalytical method validation and to establish procedures for validation. Key topics included acceptable practices for documenting and validating methods, the importance of hyphenated mass spectrometric-based assays, ligand-based assays, high-throughput systems, and the measurement and stability of drug metabolites. The need for stereoselective determinations was also discussed. The introduction emphasizes the critical role of well-characterized and fully validated analytical methods in generating reliable data for bioavailability, bioequivalence, and pharmacokinetic studies. It highlights the ongoing advancements in technology and the need for clearly defined validation criteria, especially for hyphenated mass spectrometry techniques like LC-MS-MS. Bioanalytical method validation involves demonstrating the reliability of a method for quantifying analytes in biological matrices. The report outlines different levels of validation, including Full Validation, Partial Validation, and Cross Validation. Full Validation is necessary for the initial development and implementation of a method, for new drug entities, and for any modifications that add new analytes. Partial Validation covers modifications such as method transfers between laboratories, instrument changes, and changes in matrix or species.