Butea monosperma (Lam.) Taubert (Synonym Butea frondosa; family Fabaceae) is a popular plant used in traditional medicine in the Indian continent. The brightly orange flowers of this plant are described with antioxidant potential. The aim of this study was to develop a bio-guided fractionation methodology in order to identify antioxidant biomarkers from B. monosperma flowers which could be used to ensure quality and efficiency of cosmetic products. The hydro-alcoholic extract of B. monosperma flowers was separated using a double Centrifugal Partition Chromatography (CPC) procedure. Presence and/or absence of specific molecule families were evaluated by High Performance Thin Layer Chromatography (HPTLC). The antioxidant activity was performed on each fraction to guide fractionation step. First CPC step separated the crude extract in three fractions of different polarities. The aqueous antioxidant fraction was then subjected to a second CPC separation leading to five sub-fractions. Butrin and isobutrin were isolated from the antioxidant sub-fraction and were highlighted as potential antioxidant marker in B. monosperma flowers. Their chemical structures were confirmed by UV, MS and NMR analyses. (C) 2011 Elsevier B.V. All rights reserved.