Objective To establish an HPLC method for simultaneous contents determination of paeoniflorin, protocatechuic acid and chlorogenic acid in Penyanjing Capsule. Methods The chromatographic separation was performed on a SHIMADZU VP-ODS-C18 column (4.6 mm × 250 mm, 5 μm); the column temperature was maintained at 30 ℃; A gradient elution of acetonitrile-0.15% phosphoric acid aqueous solution was adopted at the flow rate of 1.0 mL/min; The UV detection wavelengths were at 231, 255, and 326 nm; The injection volume was 20 μL. Results Paeoniflorin, protocatechuic acid and chlorogenic acid could be separated efficiently with this condition. The regression equation was Y=1546.4128X+127.0756 (r=0.9999), Y=2925.8468X+2204.1076 (r=0.9998), Y=893.9045X-261.7315 (r=0.9994), respectively. Paeoniflorin, protocatechuic acid and chlorogenic acid were linear in the range of 150.054–1254.50 ng, 162.33–1352.75 ng, 11.43–95.25 ng, and the average recoveries were 97.60%, 102.09% and 98.52%. Conclusion The method is simple, convenient and accurate, which can be used to the quality control of Penyanjing Capsule.