The use of liquid chromatography (LC) coupled with triple quadrupole linear ion trap (Qtrap) mass spectrometry (MS) for both quantitative and qualitative analysis in drug metabolism and pharmacokinetic studies is of great interest. Here, a new Qtrap-based analytical methodology for simultaneous detection, structural characterization and semi-quantitation of in vitro oxidative metabolites and glutathione trapped reactive metabolites was reported. In the current study, combined multiple ion monitoring and multiple reaction monitoring were served as surveying scans to trigger product ion spectral acquisition of oxidative metabolites and glutathione adduct, respectively. Then, detection of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches. Additionally, on-line ultraviolet (UV) detection was employed to determine relative concentrations of major metabolites. Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and glutathione adducts, but also identified their major oxidative metabolism and bioactivation pathways. The results demonstrated that the LC/UV/MS method enabled Qtrap to perform the comprehensive profiling of oxidative metabolites and glutathione adducts in vitro.