To develop a RP-HPLC method for the determination of quercetin, luteolin, apigenin and acacetin in Flos Chrysanthemi indici. An Eclipse XDB-C18 column (4.6 mm x 250 mm, 5 microm) was used at 25 degrees C with the mobile phases of methanol-0.2% phosphatic acid in a gradient manner. The flow rate was set at 1.0 mL x min(-1). The detection wavelength was 350 nm. The linear response ranged from 1.02-20.48 mg x L(-1) for quercetin (r = 0.9994, n = 5), 1.03-20.54 mg x L(-1) for luteolin (r = 0.9992, n = 5), 1.12-22.40 mg x L(-1) for apigenin (r = 0.9995, n = 5), 1.01-20.22 mg x L(-1) for acacetin (r = 0.9998, n = 5), respectively. Recoveries were 101.3% with RSD 1.3% for quercetin, 100.62% with RSD 1.4% for luteolin, 98.42% with RSD 1.7% for apigenin and 99.02% with RSD 0.8% for acacetin. A significant difference (alpha = 0.01) among the contents of four flavonoids and total flavonoids was found. The method is quick, simple and repeatable for simultaneous determination of quercetin, luteolin, apigenin and acacetin in Flos Chrysanthemi Indici.