The use of herbal medicines has tremendously increased over the past few decades. Case reports and controlled clinical investigations of herbal-drug interactions have been reported. Since Cytochrome P450 (CYP) enzymes play an important role in drug interactions. The evaluation of the influence of herbal medicines on the activities of CYPs is beneficial to promote scientific and rational clinical use of herbal medicines.Herein, we aimed to develop and validate a method to simultaneously quantify seven CYP cocktail probe drugs consisting of phenacetin (PNC), bupropion (BPP), losartan potassium (LK), omeprazole (OMP), dextromethorphan (DM), chlorzoxazone (CZZ) and midazolam (MDZ) and their respective metabolites in a single acquisition run and use this method to evaluate the influence of Zhuanggu Guanjie Pill (ZGGJP) on seven CYPs.A cost-effective and simple UHPLC-(±)ESI-MS/MS method for simultaneous determination of seven probe drugs and metabolites in rat plasma was developed and validated. Male and female rats were randomly divided into three groups and treated with 1.2 g/kg/d ZGGJP, 5 g/kg/d ZGGJP and 0.5% CMC-Na for 14 consecutive days. After 24 h of the last administration, all rats were administrated orally with probe drugs. The influence of ZGGJP on the CYPs was carried out by comparing the metabolic ratio (Cmax, AUC0-t) of metabolites/probe drugs in rats.The calibration curves were linear, with correlation coefficient > 0.99 for seven probe drugs and their corresponding metabolites. Intra- and inter-day precisions were not greater than 15% RSD and the accuracies were within ±15% of nominal concentrations. The ZGGJP showed significant inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.ZGGJP had inductive effects on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.