The structural elucidation of metabolites of ritonavir and indinavir, HIV-protease inhibitor drugs, by liquid chromatography–electrospray ionization mass spectrometry is described. Ritonavir and indinavir were biotransformed separately by incubation with transplant quality human liver microsomes. The incubation mixture was then analyzed by HPLC coupled to ion trap (ITMS) and triple quadrupole mass analyzers. The metabolites retained most of the structural features of the parent molecules. Baseline chromatographic resolution of isobaric species by gradient elution HPLC permitted rapid structural identification of these metabolites. Both drugs were biotransformed primarily by oxidative and hydrolytic pathways to numerous metabolites that retained many of the features of the parent molecules. Triple quadrupole and ion trap mass spectrometry were applied jointly to thoroughly detect and thoroughly characterize these metabolites. Furthermore, retention-time and data-dependent scanning assured acquisition of detailed MS–MS spectra for rapid detection of metabolic pathways of ritonavir and indinavir. Comparison of the ITMS and triple quadrupole data showed qualitative and quantitative differences in the mass spectral patterns, suggesting that these instruments should be used in parallel to ensure comprehensive metabolite detection and characterization by LC–MS.