A simple, reproducible and accurate RP-LC method has been developed for simultaneous analysis of seven flavonoids (quercimeritrin, hyperoside, isoquercetrin, astragalin, quercetin, quercetin-3'-glucoside, and tiliroside) in Flos Gossypii (FG), a drug used in Xinjiang, China. The analytes were separated on a C(18) column by elution with a gradient prepared from methanol, acetonitrile, and 0.2% aqueous phosphoric acid. The method was validated for linearity, accuracy, precision, sensitivity, and system suitability. It has been successfully used for evaluation of ten samples of different origin from Xinjiang, China. A similar pattern was observed for all the FG samples analyzed; quercimeritrin, isoquercetrin, and quercetin-3'-glucoside were the major compounds. The method is a simple, rapid, and reliable tool for quality control and may help reveal the correlation between the bioactivity of the FG and individual flavonoid content.