Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules, and therefore have the potential to disrupt biological function and elicit idosyncratic adverse drug reactions. We recently found that 5-substituted 2-(alkylthio)-1, 3, 4 thiadiazole structural motif can be added to the growing list of structural alerts for bioactivation. This bioactivation pathway was first observed for a commercially available 4, 5 substituted 2-(methylthio)-1, 3, 4 thiadiazole compound. When this compound was incubated with human liver microsomes in the presence of NADPH and glutathione (GSH), an unusual GSH adduct was formed. Structure identification of the GSH adduct by high resolution accurate mass instruments indicated the replacement of alkylthio group by GSH. This specific bioactivation pathway has not been reported in the literature based on our knowledge. The possible mechanism for this GSH adduct formation could be attributed to the enzymatic oxidation of alkylthio group to form sulfoxide and sulfone, which are very good leaving groups and are readily displaced due to nucleophilic attack by GSH. We also investigated the presence of this bioactivation pathway in more than a dozen commercially available compounds containing 2-(alkylthio)-1, 3, 4 thiadiazole motifs and having different substitutions at 4 and/or 5 positions of thiadiazoles. The structural identification of the unusual GSH adducts and effect of substitutions of this bioactivation pathway will be presented in detail in this report. These results can help medicinal chemists make necessary chemical modifications and thoughtful drug design to avoid potential liability related to 2-(alkylthio)-1, 3, 4 thiadiazoles.