A simple,precise,and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2 00-O-glucoside,vitexin-2 00-O-rhamnoside,rutin,vitexin,and hyperoside.The HPLC separation was performed using a Shim-pack VP-ODS C 18 column(250 mm 4.6 mm i.d.,5 mm) with the isocratic mobile phase consisting of tetrahydrofuran/acetonitrile/0.05% phosphoric acid solution(20:3:77,v/v/v),and the flow rate was set at 1.0 mL/min.UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min.The method was linear in the range of 4.12-206.00 mg/mL for vitexin-2 00-O-glucoside,4.05-202.50 mg/mL for vitexin-2 00-Orhamnoside,1.64-82.00 mg/mL for rutin,1.74-87.00 mg/mL for vitexin,and 1.41-70.60 mg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998.The limit of detection(LOD) and limit of quantitation(LOQ) were 0.6 and 2 ng for vitexin-2 00-O-glucoside,0.6 and 2 ng for vitexin-2 00-O-rhamnoside,0.3 and 1 ng for rutin,1 and 3 ng for vitexin,and 0.5 and 2 ng for hyperoside,respectively.Intra-and inter-day precision and accuracy(RSD) were less than 3%.The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves,hawthorn fruits,and the preparations containing hawthorn leaves or fruits. A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2 00-O-glucoside, vitexin-2 00-0- rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C18 column (250 mm 4.6 mm id, 5 mm) with the isocratic mobile phase consisting of tetrahydrofuran / acetonitrile / 0.05% phosphoric acid solution (20: 3: v), and the flow rate was set at 1.0 mL / min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12-206.00 mg / mL for vitexin -200-O-glucoside, 4.05-202.50 mg / mL for vitexin-2 00-Orhamnoside, 1.64-82.00 mg / mL for rutin, 1.74-87.00 mg / mL for vitexin, and 1.41-70.60 mg / mL for hyperoside with the correlation coefficient for each analyte more than 0.998. limit of detection (LOD) and limit of quantitation (LOQ) were 0.6 and 2 ng for vitexin-2 00-0-glucoside, 0.6 and 2 ng for vitexin-2 00-0 -rhamn oside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Intra-and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.