An high performance liquid chromatographic method for the quantitative determination of quercetin-3-O-β-D-glucuronide and quercetin in Polygonum perfoliatum L.was developed.The separation was performed on a Lichrosher 5-C18 column(250 mm × 4.6 mm,5 μm) together with a C18 guard column by gradient elution using methanol-0.05% phosphoric acid as mobile phase at a flow rate of 1 mL/min.The detection wavelength was set at 258 nm and the colume temperature was 25 ℃.Two compounds were successfully separated in 30 min.The calibration curves were linear in the ranges of 0.037 6-3.920 0 mg for quercetin-3-O-β-D-glucuronide and 0.004 2-0.440 0 mg for quercetin.The limits of detection(LOD) and limits of quantitation(LOQ) were 2.509,0.282 ng and 6.272,0.702 ng，respectively.The mean recoveries of two analytes were 99% and 98%,respectively.The established method was applied in the determination of contents of both two compounds in 15 samples obtained from different regions,and the results showed that the proportion of both compounds in different samples were great different.The proposed method is simple,rapid and accurate,and can be used for the quality control of Polygonum perfoliatum L..