This study employed the online HPLC-2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate radical cation (ABTS(+)) bioassay to rapidly determine antioxidant compounds occurring in the licorice extract of Glycyrrhiza uralensis. The negative peaks of the ABTS(+) radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS(+)-based antioxidant activity profile showed that three peaks exhibited antioxidant activity, and then the high-speed counter-current chromatography technique of preparative scale was successfully applied to separate the three peaks I-III in one step from the licorice extract. The high-speed counter-current chromatography was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (6.5:5.5:6:4, v/v). Yields of the three peaks, dehydroglyasperin C (I, 95.1% purity), dehydroglyasperin D (II, 96.2% purity), and isoangustone A (III, 99.5% purity), obtained were 10.33, 10.43, and 6.7% respectively. Chemical structures of the purified dehydroglyasperin C (I), dehydroglyasperin D (II), and isoangustone A (III) were identified by ESI-MS and (1)H- and (13)C-NMR analysis.