Abstract: OBJECTIVE To establish a UHPLC-MS/MS method for simultaneously determination of phenacetin, tolbutamide, omeprazole, metoprolol and midazolam in rat plasma. METHODS The plasma sample was prepared by acetonitrile precipitation. The analytical column was Agilent ZORBAX Eclipse Plus C₁₈(2.1 mm×50 mm, 1.8 μm). The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile with gradient elution. The flow rate was 0.4 mL·min^-1. Detection equipped with electrospray ionization source with multiple reaction monitoring. The tandem mass ion transitions monitored were m/z 180.1→109.9 for phenacetin(M+H⁺), m/z 271.1→91.0 for tolbutamide, m/z 346.1→135.9 for omeprazole (M+H⁺), m/z 268.2→115.0 for metoprolol(M+H⁺), m/z 326.1→290.8 for midazolam(M+H⁺), and m/z 237.1→194.0 for carbamazepine(IS,M+H⁺). Blood sample was collected of the tail vein at multiple time point after a single oral dose of administration of phenacetin(10 mg·kg^-1), tolbutamide (1 mg·kg^-1), omeprazole(10 mg·kg^-1), metoprolol(10 mg·kg^-1) and midazolam(10 mg·kg^-1) in six male SD rats. The pharmacokinetic parameters were calculated by DAS. RESULTS The calibration curve was linear over the range of curve for the five probe drugs. Intra-day and inter-day RSDs for assaying the plasma sample were both <15%, the recovery rate was >75% for the five drugs. The phenacetin AUC_0-t was (5 868.30±2 062.87) ng·mL^-1·h; the tolbutamide AUC_0-twas (58 056.34±15 569.16) ng·mL^-1·h; the omeprazole AUC_0-t was (14 181.67±4 085.40) ng·mL^-1·h; the metoprolol AUC_0-t was (1 123.67±180.469) ng·mL^-1·h; the midazolam AUC_0-twas (946.91±322.03) ng·mL^-1·h. CONCLUSION The methods is proved to be sensitivity, simple and accuracy, and can be used as an analytical method for the study of CYP450 enzyme activity and related research.