The metabolic bioactivation of a drug to a reactive metabolite (RM) and its covalent binding to cellular macromolecules is believed to be involved in clinical adverse events, including idiosyncratic drug toxicities. Therefore, it is important to assess the potential of drug candidates to generate RMs and form drug-protein covalent adducts during lead optimization processes. In this study, the RM formation of some marketed drugs were quantitatively assessed by means of a sensitive and robust detection method that we have established using (35)S-glutathione ((35)S-GSH) as a trapping agent. Problematic drugs well-known to generate RMs exhibited a relatively high rate of (35)S-GS-adducts to RM (RM-GS) formation, which contrasted with safe drugs. For practical use in lead optimization processes, a series of new chemical entities were tested and hints on the structural modifications needed in order to minimize their RM formation were provided. Furthermore, the RM-GS formation rates of a number of compounds were compared using their in vitro covalent binding yields to liver proteins determined with (14)C-labeled compounds, demonstrating that the RM-GS formation rate could be a substitute for the covalent binding yield within the same series of compounds.