Characterization of metabolites and understanding metabolic soft-spots in a chemical scaffold is routinely carried out in drug discovery in liver microsomal models across multiple species. However, oxidative metabolites identified in microsomal incubations in the presence of NADPH may arise from either CYP or FMO enzymes. The objective of our investigation was to evaluate the efficiency of commercial, irreversible, non-selective CYP inhibitors, either individually or in combination with one another, for application in biotransformation studies. Buspirone, is largely metabolized by CYPs and the metabolites have been well characterized. Therefore it was chosen as a probe substrate. Known time dependent CYP inhibitors such as 1-aminobenzotriazole, (1-ABT), proadifen or 2-(diethylamino)ethyl 2,2-diphenylpentanoate (SKF525A) and a cocktail (I-cocktail) of known specific irreversible CYP inhibitors were evaluated for their CYP knockout efficiencies. The I-cocktail consisted of furafylline (CYP1A2), 8-methoxypsoralen (CYP2A6), ticlopidine (CYP2B6), amiodarone (CYP2C8), tienilic acid (CYP2C9), ticlopidine (CYP2C19), paroxetine (CYP2D6), diethylthiocarbamate (CYP2E1) and erythromycin (CYP3A4). The experiments were carried out using rat and human liver microsomes fortified with NADPH with and without mechanism based inhibitor pre-treatment. Metabolite(s) identification of buspirone was carried out on a Thermo-Finnigan LTQ-Orbitrap-XL® with accurate mass measurement and MSn capabilities. Control experiments revealed that less than 5% buspirone remained at the end of the one hour incubation period, in contrast to 89%, 69% and 77% remaining for 1-ABT, SKF525A and I-cocktail pre-treatments respectively in human liver microsomes. Formation of an N-dealkylated carboxylic acid metabolite was inhibited in all three pre-treatments. Another metabolite formed subsequent to piperazine ring opening was absent in human liver microsomal incubations pre-treated with I-cocktail, although present in reduced peak areas in the incubations pre-treated with 1-ABT and SKF525A. Effects of the above time dependent inhibitors on each of the fourteen or more identified buspirone metabolites in rat and human liver will be presented.