Abstract: The effects of Scrophularia ningpoensis on the activities of 4 Cytochrome P450 enzymes (CYP3A4, CYP2D6, CYP2C9 and CYP1A2) in rats was investigated using the Cocktail probe drug method. Twenty-four healthy male Sprague-Dawley rats were randomly divided into three groups: group A (multiple-doses of 150 mg/kg Scrophularia ningpoensis for 7 d), group B (single-dose of 150 mg/kg Scrophularia ningpoensis), and group C (control group). To each group after administering Scrophularia ningpoensis for 30 min, the mixture solution of probe drugs midazolam (3 mg/kg, evaluation of CYP3A4 activity), metoprolol (20 mg/kg, evaluation of CYP2D6 activity), losartan (5 mg/kg, evaluation of CYP2C9 activity) and phenacetin (5 mg/kg, evaluation of CYP1A2 activity) were administered orally on the 7^th d. Rat plasma samples were collected at the arranged time points, and the plasma concentrations of 4 probe drugs in the samples were simultaneously determined using the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method after processing. The results showed that Scrophularia ningpoensis had almost no significant effect on the pharmacokinetic parameters of losartan and phenacetin. However, a single-dose of Scrophularia ningpoensis induced a slow metabolism of midazolam. In addition, the pharmacokinetics of metoprolol was affected significantly by the multiple doses of Scrophularia ningpoensis, which induced rapid metabolism. The results suggested that Scrophularia ningpoensis has no significant effect on the enzymatic activities of CYP2C9 and CYP1A2, but it might inhibit enzymatic activities of CYP3A4 or induce CYP2D6.