Objective: To establish an HPLC method for the simultaneous determination of five active components (paeoniflorin, liquiritin, ammonium glycyrrhizinate, edomin and osthole)in Pifukang lotion with multiple UV wavelengths.Methods: A Diamonsil C18 (200 mm×4.6 mm,5 μm) column was used with the mobile phase consisting of 0.1% hydrochloric acid and acetonitrile with gradient elution.The flow rate was 1.5 ml·min-1, and the column temperature was 30℃.Paeoniflorin was detected at 232 nm(0-6 min), liquiritin was detected at 277 nm(6-10 min),and ammonium glycyrrhizinate, edomin and osthole were detected at 254 nm(after 10 min).Results: The linear range of paeoniflorin,liquiritin,ammonium glycyrrhizinate, edomin and osthole was 28.200-282.000 μg·mL-1(r=0.999 7),12.150-121.500 μg·ml-1(r=0.998 8),13.420-134.200 μg·ml-1(r=0.999 5),0.047-0.466 μg·ml-1(r=0.999 9) and 2.38-23.8 μg·ml-1(r=0.999 9), respectively.The average recovery (RSD) was 98.49%(0.71%), 99.00%(0.62%), 98.38%(0.85%), 97.36%(0.92%) and 97.70%(0.78%), respectively (n=6).Conclusion: The established method is simple, accurate, highly sensitive and well reproducible, which can be used for the determination of the five active ingredients in Pifukang lotion.