Sesquiterpenes lactones including costunolide and dehydrocostus lactone, were isolated from roots, which had exhibited a wide range of biological activities such as anti-cancer, anti-inflammatory, antiulcer, and immunomodulatory activities. High-speed counter-current chromatography (HSCCC) was applied for the rapid preparative isolation of sesquiterpenes lactones from roots. A solvent optimization method for HSCCC was presented, i.e. the separation factors of compounds after the partition coefficient (K) of solvent system should be investigated. Using this method, 150 mg of costunolide, 140 mg of dehydrocostus and 15 mg of 10-methoxy-artemisinic acid with purities of 95%, 98%, and 98% were obtained within 150 min. The structures of three compounds were identified by mass spectrum (MS) and nuclear magnetic resonance (NMR) spectroscopy. These results offered an efficient strategy for separation of potentially health-relevant phytochemicals from roots.