Abstract: A gradient high performance liquid chromatography (HPLC) coupled with UV-vis photodiode array detection (DAD) was developed for the separation of rutin, hyperoside, and quercetin from the flavoind extracts from hawthorn powder.The flavoinds were extracted using the solvent of 70% ethanol (by volume) .Chromatographic separation was performed in DevelosilC30 column (250mm×4.6mm ID, 5μm) with an eluent consisting of acetonitrile and 0.4% Phosphate at a flow rate of 1.0mL/min, the eluate was monitored at 360nm.The result showed that separation and identification of rutin, hyperoside, quercetin, and other components were completed in 30min.The linear ranges of rutin, hyperoside, and quercetin were 0.0050~0.1600mg/mL (R2=0.9999) , 0.0016~0.0500mg/mL (R2=0.9996) and 0.0005~0.0150mg/mL (R2=0.9997) , respectively.This method was precise and accurate (RSD 0.48%~0.78%) , the average recoveries ranged from 92.36% to 98.64%, and can be used for the precise quantification of the three flavoinds and other flavoinds of dry plant material such as hawthorn power.