A combinative method using DPPH-HPLC and HSCCC has been developed to screen and separate antioxidants from ethyl acetate fraction of Pueraria lobata flower. Under the target-guidance of DPPH-HPLC experiment, 18 isoflavones were isolated by HSCCC using several elution modes, such as classical elution, stepwise elution, extrusion elution and recycling elution. Following an additional dean-up step by crystallization, the purities of all the antioxidant compounds were all over 98.0% as determined by HPLC, and their structures were elucidated by UV, MS/MS and NMR analysis. Compounds glycitin (2), tectoridin (4), daidzin (10), 3'-hydroxydaidzein (13) and tectorigenin (14) were found to be the major antioxidants in P. lobata flower based on the relative peak areas in the HPLC chromatogram. Ten compounds including one new compound were first isolated from P. lobata flower. New compound, irisolidone-7-O-beta-D-glucopyranpsyl-(1 -> 6)-beta-D-glucopyranoside (6), showed moderate DPPH antioxidant activity with IC50 value of 110.3 mu g/ml. Results of the present study indicated that the combinative method using DPPH-HPLC and HSCCC was a speedy, efficient and reproductive technique to systematically isolate antioxidant compounds from complex natural products. (c) 2012 Elsevier B.V. All rights reserved.