High-performance liquid chromatography coupled to an on-line ABTS+-based assay (on-line HPLC-ABTS+) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2mg of myricetin, 28.9mg of quercetin, and 10.6mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.