Objective To establish a preparative method for rupestonic acid reference substance from Artemisia rupestris by HPLC. Methods After raw material being extracted with 95% ethanol, the ethanol extracts were evaporated to dryness under reduced pressure. Then the dry powder was extracted by ethyl acetate, the ethyl acetate extract was fractionated by column chromatography on silica gel (100200 mesh.) using petroleumbenzine-ethyl acetate (3 ! 1) as the eluent. The collected fraction was purified by preparative HPLC Results The purity of the product quantitated by normality was over 98%. t-onclusion The developed method is simple and rapid and the product of rupestonic acid can be used as reference substance.