AbstractIntroductionIn Eastern Europe, especially Russia, the fruiting body of Inonotus obliquus has been used as a folk medicine for cancer since the sixteenth or seventeenth century. Inotodiol and trametenolic acid are considered to be the main bioactive compounds of the fruiting body of the mushroom. These compounds show various biological activities, including anti‐tumour, anti‐viral, hypoglycaemic, anti‐oxidant and cyto‐protective. However, effective methods for isolating and purifying inotodiol and trametenolic acid from the fruiting body of Inonotus obliquus are not currently available.ObjectiveTo develop a suitable preparative method in order to isolate inotodiol and trametenolic acid from a complex Inonotus obliquus extract by preparative high‐speed counter‐current chromatography (HSCCC).MethodologyInotodiol and trametenolic acid were rapidly isolated and purified from the chloroform extract of Inonotus obliquus (Fr.) by HSCCC with evaporative light scatting detection (ELSD). The purity of the obtained target compounds was analysed by high‐performance liquid chromatography (HPLC) with ELSD. The structures of the two compounds were identified by 1 H NMR and 13 C NMR.ResultThe target compounds were finally isolated and purified with a solvent system of hexane:ethyl acetate:methanol:water (1:0.4:1:0.4, v/v/v/v). In a single operation, 100 mg of the I. obliquus extracts yielded 13.0 mg of inotodiol and 7.0 mg of trametenolic acid. The entire separation and purification process took less than 5 h. The purities of obtained inotodiol and trametenolic acid were 97.51 and 94.04%, respectively.ConclusionHSCCC‐ELSD was an efficient and rapid method for the separation and purification of inotodiol and trametenolic acid from I. obliquus. Copyright © 2011 John Wiley & Sons, Ltd.