Objective This study aimed to characterize the effects of bovine serum albumin (BSA) on 16 specific probe reactions by nine human drug-metabolizing enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4) in vitro.Methods A11 incubations were performed on pooled human liver microsomes (HLM) in the absence or presence of BSA (0.5%, 1%, 2%, w/v) for 0, 10, 20, 30, 45 and 60 min.Phenacetin O-deethylation (CYP1A2), coumarin-7-hydroxylation (CYP2A6), bupropion 2-hydroxylation (CYP2B6), taxol 6-hydroxylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), tolbutamide methyl-hydroxylation (CYP2C9),S-mephenytoin 4-hydroxylation (CYP2C19), omeprazole 5-hydroxylation (CYP2C19), midazolam 4-hydroxylation (CYP2C19),(±)-bufuralol 1-hydroxylation (CYP2D6), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1),midazolam 1-hydroxylation (CYP3A4), testosterone 6β-hydroxylation (CYP3A4), nifedipine oxidation (CYP3A4), omeprazole sulfonylation (CYP3A4) were used as the probes.These incubations were terminated by the addition of ice-cold acetonitrile, and then centrifuged to get the supematant layer for the LC/MS-MS analysis by API 4000.All data were analyed by using GraphPad Prism 5.0 software.Results By comparison the generation of probe metabolites to the incubation time with or without BSA in the incubation mixture, we found that there were different types of the influence by the addition of BSA to the probe reaction.Firstly, for the probes of (±)-bufuralol, dextromethorphan, coumarin, tolbutamide, taxol, phenacetin, bupropion, omeprazole (5-hydroxylation), S-mephenytoin,the conversion rate catalyzed by specific CYP enzyme was enhanced in the presence of BSA apparently, with the first five probes show a concentration dependence of increase with BSA (0.5%, 1%, 2%, w/v).Secondly, for the probes of testosterone, midazolarn (l-hydroxylation and 4-hydroxylation), diclofenac, nifedipine etc., the rate of conversion was significantly decreased in the presence of BSA.Thirdly, for the probes ofomeprazole (sulfonylation) and chlorzoxazone, there was no significant difference in the conversion with or without the BSA in the incubation.Conclusion The effects of BSA on human liver cytochromes activity vary with the specific probe reactions for the corresponding enzymes.