Cocktail probe drug method was used to evaluate the effect of cold treatment drugs such as Gegen Decoction on cytochrome P450 (CYP450) enzyme activity in rat liver. The in vitro incubation system of rat liver microsomes was optimized. Based on LC tandem mass spectrometry (LC-MS/MS), an analytical method for simultaneous determination of the content of metabolites of each subenzyme probe in rat liver microsome CYP450 enzyme was established. Cocktail probe drug method was used to characterize the effects of Gegen Decoction, a classical prescription for the treatment of wind-cold cold, and acetaminophen, chlorpheniramine maleate, anhydrous caffeine and nacotine hydrochloride, which were commonly used in the treatment of cold, on the activity of CYP2D6, CYP2C9, CYP3A4 and other sub-enzymes in rat liver microsomes, so as to provide reference for the safety of clinical drug combination. The results showed that the established analysis method was stable and reliable, which met the requirements of biological sample determination. After optimization, the protein concentration of rat liver microsomes in the incubation system was 3.00 mg·mL^-1, the incubation time was 240 min, and the terminator was acetonitrile. The K_m values of three CYP subenzyme specific probe substrates dextromethorphan, testosterone and tolbutamide were 21.49, 87.33 and 354.7 μmol·L^-1, respectively. The effectiveness of the established incubation system was verified by positive inhibitors. Compared with the blank control group, when the concentration of each Western medicine and the concentration of Gegen Decoction extract were within 100.00 μmol·L^-1 and 3.00 mg·mL^-1, respectively, Gegen Decoction extract, chlorpheniramine maleate and noscapine hydrochloride had inhibitory effects on CYP2C9 and CYP2D6 enzymes in rat liver microsomes; anhydrous caffeine had inhibitory effects on CYP2C9 and CYP3A4 enzymes in rat liver microsomes; no significant inhibitory effect on acetaminophen; the induction effect of narcotine hydrochloride on CYP3A4 enzyme in rat liver microsomes was greater than 40% of the activity of positive inducer, suggesting that there may be a certain risk of drug interaction when combined with narcotine hydrochloride, and attention should be paid to the dose of drug treatment. The experimental protocol strictly adhered to the guidelines of the Ethics Committee of Animal Research of Guangdong Pharmaceutical University (Approval: gdpulacspf2022137).