An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450(CYP) enzymes.This method employed a cocktail of six probe substrates(i.e.,phenacetin.amodiaquine.diclofenac,S-mephenytoin,dextromethorphan and midazolam for CYP1A2,2C8.2C9,2C19,2D6 and 3A4.respectively) as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions.The corresponding marker metabolites(i.e.,acetaminophen,N-deselhylamodiaquine,4-OH-diclofenac.4-OH-Smephenytoin,dextrorphan and 1-OH-midazolam) in the incubates were quantified using LC-MS/MS methods either by an internal standaid(IS) calibration curve or a simplified analyte-to-IS peak area ratio approach.The results showed that the IC_(50) values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature.Besides,no remarkable difference was observed between the two quantification approaches.In conclusion,this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition. An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP) enzymes. This method employed a cocktail of six probe substrates (ie, phenacetin.amodiaquine.diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8.2C9, 2C19, 2D6 and 3A4.respectively) as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (ie, acetaminophen, N-deselhylamodiaquine, 4-OH-diclofenac.4-OH-Smephenytoin, dextrorphan and 1-OH-midazolam) in the incubates were quantified using LC-MS / MS methods either by an internal standaid (IS) calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC 50 (50) values ​​determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature.B esides, no remarkable difference was observed between the two quantification approaches. conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.