Abstract: OBJECTIVE To develop a high performance liquid chromatography method for the determination of the activity of CYP2C9 in rat liver microsomes using diclofenac as probe drug. METHODS The analytical column was packed with Agilent ZORBAX SB-C₁₈. The mobile phase was acetonitrile-water-0.1% trifluoroacetic acid using gradient elution and the flow rate was 1.0 mL·min^-1. The UV detection wavelength was 278 nm. The incubation was performed with diclofenac at 37 ℃ for 30 min, then the reaction was terminated by adding HCl solution and ethyl acetate. Then diazepam solution was added. After vortexing for 2 min, the samples were centrifuged at 13 000 r·min^-1 for 5 min. The organic phase was transferred into a clean tube and evaporated under nitrogen stream. The residue was dissolved in 100 μL of mobile phase. Then a 20 μL aliquot of the solution was injected into the HPLC system for analysis. RESULTS Diclofenac, 4-hydroxydiclofenac and diazepam were perfectly separated. Excellent liner relationship of 4-hydroxydiclofenac was obtained from 0.05-10.0 μmol·L^-1(r=0.999 8). RSD of inter- and intra-day preasion were <10% for diclofenac and 4-hydroxy diclofenac, and the method recoveries were >75%. The HCl and ethylacetate were selected as reagent to terminate the reaction. Km was 26.87 μmol·L^-1 and Vmax was 2.359 nmol·min^-1·mg^-1 pro. CONCLUSION The method is steady, accurate and suitable for assaying CYP2C9 activity which can be used to evaluate the pharmacokinetics of CYP2C9 in rat liver microsomes.