The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM incubations. In addition, clomipramine and mefenamic acid that have the frame structures susceptible to P450-mediated bioactivation were investigated. As a result, the MRM-EPI analysis revealed multiple GSH adducts of clomipramine and mefenamic acid in HLM incubations possibly mediated by epoxide and/or quinone imine intermediates. Compared with the neutral loss (NL) and precursor ion (PI) scanning analysis, the MRM-based approach provided superior sensitivity and selectivity for GSH adducts. It also enabled the sensitive acquisition of EPI spectra with rich fragmentation in the same LC/MS run, which were useful for the rapid structure elucidation of GSH adducts and the elimination of false positives. The MRM-EPI experiment can be employed for high throughput screening of reactive metabolites and should be especially applicable to compounds of the same chemotype. Also, it can be applied in conjunction with the PI or NL scan as a comprehensive method for the analysis of reactive metabolites in a drug discovery setting.