This study examined the efficacy of four common in vitro assay systems in producing metabolic profiles consistent with in vivo data for drugs of abuse. Cocaine (COC) was selected for this study because of its complex biotransformation pathways, diverse metabolic processes and because extensive Phase I and Phase II metabolomic examination of COC has not yet been reported by means of in vitro assay. COC metabolism was assessed with a series of common in vitro assay systems (human liver microsomes, cytosol and human liver S9 fraction and horseradish peroxidase) using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring. Qualitative and quantitative differences in analyte production were noted among the various active Phase I and Phase II metabolic systems. Assay incubation time was found to be a determining factor in metabolic profile, specifically with primary versus secondary metabolite formation. Regioselective arene hydroxylation of COC was conclusively documented in human hepatic metabolic models, while peroxidase-based assay systems displayed less selectivity in oxidative aryl biotransformation. Results demonstrate the applicability of in vitro systems in studying COC metabolite production and the impact of assay selection and variation in method parameters on metabolite profiles for this important drug of abuse.