Abstract: AIMTo establish an HPLC method for simultaneous determination of four flavonoids in Hypericum perforatum L., ruitn, hyperin, avicularin and quercetin. METHODSC₁₈ column was used, the chromatography was carried out with a linear gradient program. The mobile phase was A: H₂O (pH 3.0～3.5 adjusted with phosphoric acid) and B: acetonitrile, at flow rate of 1.0 mL·min^-1, peaks were detected at 254 nm. RESULTS The linear range of rutin was 1.376～8.256 μg·mL^-1 (γ=0.9999), hyperin 3.160～18.960 μg·mL^-1 (γ=0.9996), avicularin 0.968～5.808 μg·mL^-1 (γ=0.9998) and quercetin 0.776～4.656 μg·mL^-1 (γ=0.9993). The average recovery of rutin was 97.8%, RSD 4.8% (n=3), hyperin 100.7%, RSD 3.7% (n=3), avicularin 97.3%, RSD 0.8% (n=3) and quercetin 100.5%, RSD 4.4% (n=3). All of RSDs of precision were less than 2% (n=5), reproducibilities less than 3%. CONCLUSIONThe method is simple, effective and feasible. It can be used to determine flavonodis in Hypericum perforatum.