Abstract: To establish an HPLC-MS/MS methods for the simultaneous determination of seven metabolites (acetaminophen,4″-hydroxymephenytoin,dextrorphan,1-hydroxymidazolam,6β-hydroxyltestosterone,4-hydroxytolbutamide,6-hydroxychlorzoxazone) of CYP450 enzymes probe substrates in rat liver microsomes,and to investigate the inhibitory potency of bencycloquidium bromide on rat liver cytochrome P450 using probe substrates method.The probe substrate (phenacetin,S-mephenytoin,tolbutamide,dextromethorphan,chlorzoxazone,midazolam or testosterone) was incubated with RLM,bencycloquidium bromide or positive control,respectively.The precipitated samples for each probe substrate were mixed and simultaneous analyzed by LC-MS/MS.The concentrations of metabolites were assayed and used to calculate the IC₅₀ which was used to evaluate the inhibition potency.The method was validated by incubating known CYP inhibitions -α-naphthoflavone (CYP1A2),omeprazole (CYP2C11),ketoconazole (CYP2C6,2E1,3A1/2),quinidine (CYP2D1/2) with individual probe substrate and rat liver microsomes,respectively.Positive controls show inhibition effects on each CYP 450s.The IC₅₀ values of bencycloquidium bromide for CYP1A2,CYP2C11,CYP2C6,CYP2E1 and CYP3A1/2 were lower than 100 μmol/L,while the IC₅₀ of bencycloquidium bromide for CYP2D1/2 was (2.16±0.22) μmol/L.It indicates that bencycloquidium bromid shows inhibitory effect on CYP2D1/2,but minor inhibitory effects of CYP1A2,CYP2C11,CYP2E1 and CYP3A1/2 in this study.