A rapid and specific reversed-phase high performance liquid chromatography (RP-HPLC) method with diode array detection (DAD) at room temperature was used and validated for the simultaneous determination of five flavonoids (catechin, CA; rutin, RU; quercetin, QU; kaempferol, KA; isorhamnetin, IS) in the extract of sea buckthorn (Hippophae rhamnoides L.) leaves. The sample pretreatment process involved ultrasonic extraction with 85% ethanol under the frequency of 80 kHz, at a temperature of 45 degrees C for 30 min and with the ratio of liquor to material of 15 mL g-1, followed by separation on HIQ SIL C18V column with methanol-acetonitrile-water (40:15:45, v/v/v) containing 1.0% acetic acid as a mobile phase. The extract was detected by DAD at the wavelength of 279 nm for CA, 257 nm for RU, 368 nm for QU, KA and IS. Calibration curves were found to be linear with the ranges of 0.011-0.520 mg ml-1 (CA), 0.007-0.500 mg ml-1 (RU), 0.019-0.280 mg ml-1 (QU), 0.010-0.440 mg ml-1 (KA) and 0.008-0.400 mg ml-1 (IS). The correlation coefficients of linear regression analysis and detection limits were between 0.9963-0.9999 and 0.00079-0.00290 mg ml-1. The contents of CA, RU, QU, KA and IS in sea buckthorn leaves were successfully determined with 3.8, 5.2, 7.3, 10.9 and 11.9 min with satisfactory reproducibility and recovery. Recoveries of the five flavonoids were between 97.27 and 99.98%. The method was applied to the determination of flavonoids in sea buckthorn leaves and was found to be simple, rapid and efficient.