| Summary: | A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from <em>Eupatorium lindleyanum</em> DC. The two-phase solvent system composed of <em>n</em>-hexane–ethyl acetate–methanol–water (1:4:2:3, v/v/v/v) was selected. From 540 mg of the <em>n</em>-butanol fraction of <em>Eupatorium lindleyanum </em>DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxy-tigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and <sup>1</sup>H-NMR.
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