Abstract: OBJECTIVE To develop a determination method of rutin, isoquercitrin, kaempferol-3-O-rutinoside, quercetin, and kaempferol in Tetrastigma hemsleyanum by HPLC. METHODS HPLC assay was carried out using an Agilent ZORBAX SB-C₁₈ column (250 mm×4.6 mm, 5 μm)with column temperature at 35℃. The mobile phase was acetonitrile-0.2% phosphoric acid in gradient elution at a flow rate of 1.0 mL·min^-1. And the detection wavelength was 360 nm. RESULTS The linear ranges of rutin, isoquercitrin, kaempferol-3-O-rutinoside, quercetin, kaempferol, and baicalin were 0.087 8-0.878 μg, 0.069 6-0.696 μg, 0.162 9-1.629 μg, 0.002 7-0.027 μg, 0.007 23-0.072 3 μg, respectively (r ≥ 0.999 7), and the average recoveries of three components were 97.44%, 96.88%, 97.00%, 96.42%, 96.24%, respectively. CONCLUSION The method is simple, quick and accurate, can be used for the quality control of the Tetrastigma hemsleyanum.