Toxicities of sulfur-based drugs have been attributed to formation of highly reactive sulfur oxo-acids and depletion of glutathione by the formation of reactive metabolites. Metabolic activation of these sulfur centers to conceivably toxic reactive metabolites (RMs) that can covalently modify proteins is considered the initial step in drug-induced toxicity. Despite considerable effort and research, detection and characterization of these RMs during drug development and therapy remains a challenge. Methimazole (MMI) and 6-propyl-2-thiouracil (PTU) are two commonly used antithyroid, sulfur-based drugs. Though effective, these drugs are associated with idiosyncratic toxicity. PTU has acquired a black box warning and physicians are calling for its withdrawal. RMs resulting from bioactivation of these drugs have been implicated in the aforementioned adverse reactions. Unfortunately, isolating and detecting RMs using traditional analytical techniques has not been successful due to their high reactivity and short life span, typically less than a minute. Current approaches in drug metabolism studies use microsomal incubations to generate RMs, which are then trapped using nucleophiles. Antithyroid drugs, however, are known to deactivate enzymes involved in their oxidation. Moreover, due to the complex nature of biological matrices and low abundance of possible toxic conjugates, this technique results in poor selectivity and sensitivity. This study developed and optimized an analytical method based on coupling electrochemical redox reactions and mass spectrometry to generate, detect and identify RMs from antithyroid drugs. The metabolites were also compared to those that were generated using chemical oxidants and I would also want to thank the members of my dissertation committee: Drs.