Radix Sophorae Tonkinensis (S. tonkinensis) is the processed lateral root of Sophora subprostrata (Leguminosae) that widely distributed over the southwest China. Radix Sophorae Tonkinensis has been widely used as a Chinese medicinal herb for the treatment of disease such as jaundice, inflammation, and aches. Herein, in order to investigate the effects of Radix Sophorae Tonkinensis on the metabolic capacity of rat cytochrome P450 (CYP) enzymes, we employed a cocktail method to evaluate the activities of CYP1A2, CYP2D6, CYP3A4, CYP2C19, CYP2C9 and CYP2B6. The experimental rats were randomly divided into two groups (control group and Radix Sophorae Tonkinensis treated group). The Radix Sophorae Tonkinensis treated group rats were given 5 g/kg Radix Sophorae Tonkinensis by continuous intragastric administration for 14 days. The mixture of six probes (phenacetin, metroprolol, midazolam, omeprazole, tolbutamide and bupropion) was given to rats by intragastric administration. The concentrations of probe drugs in rat plasma were measured by UPLC-MS/MS. The results showed that continuous intragastric administration for 14 days may inhibit the activities of rat CYP450 isoforms CYP2D6, CYP2C19 and CYP2B6. This finding may provide guidance for rational clinical uses of Radix Sophorae Tonkinensis.