OBJECTIVE To develop a RP-HPLC method for simultaneous determination of orientin, isorientin, vitexin and isovitexin in Lophatherum gracile from different habitat and harvesting time. METHODS The HPLC method was applied and the chromatographic column was a Waters XBridge C18 column (4.6 mm x 250 mm, 5 microm). The mobile phase consisted of methanol-0.05% acetic acid (35:65). The flow rate was 1.0 mL min(-1) and the detection wavelength was set at 340 nm. The column temperature was set at 25 degrees C. RESULTS Four components were isolated well, the linear relationships were excellent. The mean recoveries and RSD values of orientin, isorientin, vitexin and isovitexin were 103.2%, 2.1%; 101.6%, 2.7%; 98.4%, 2.3%; 99.2%, 1.8%, respectively. CONCLUSIONS The HPLC method is simple, sensitive and reliable, and can be used for the quality control of the medicinal material.