To develop a method for separation and quantitative determination of four flavonoids in Tetrastigma hemsleyanum.The Separation was performed on a Waters Preparative scale LC-MS autopurification system. Several spectral analyses, such as NMR,HR-MS and UV were used to identify the chemical structures. Quantitative assay was performed on a UHPLC Phenomenex Kinetex C18 column (100 mm x 4.6 mm, 2.6 µm) with the mobile phase consisting of acetonitrile( B) and water containing 0. 1% formic acid (A) in a gradient mode at a flow rate of 0.4 mL/min. The column temperature was at 30 °C and the optimum detection wavelength of DAD was set at 350 nm.Four flavonoids were isolated from the root of Tetrastigma hemsleyanum, which were identified as rutin, isoquercitrin, kaempferol-3-O-rutinoside and astragalin. As to quantitative analysis, a good separation of four flavonoids with in 17.5 min, the linear range of rutin, isoquercitrin, kaempferol-3-O-rutinoside and astragalin was 0.283-11.32 µg/mL (r1 = 0.9998), 0.311-12.44 µg/mL (r2 = 0.9994), 0.277-11.08 µg/mL (r3 = 0.9995) and 0.103-2.06 µg/mL (r4 = 0.9990), respectively. The average recovery (n = 6) was 99.75% (RSD = 2.15%), 98.73% (RSD = 2.58%) ,98. 03% (RSD = 2.23%), and 97.62% (RSD = 1.95%), respectively.Four flavonoids are isolated from the root of Tetrastigma hemsleyanum for the first time, and the UHPLC method is simple, rapid and accurate, which can be used for quantitative analysis of multi-component of Tetrastigma hemsleyanum and provide a novel approach for evaluation of the quality of Tetrastigma hemsleyanum comprehensively.