A high-performance liquid chromatographic method for the determination of dehydrotumulosic acid in plasma of rats having been administrated orally with the traditional Chinese medicinal preparation Ling-Gui-Zhu-Gan decoction was developed. Plasma samples taken from rats were acidified with hydrochloric acid and extracted with ethyl acetate. Separation of the main effective constituent dehydrotumulosic acid was accomplished on a C(18) stationary phase and a mobile phase of methanol-acetonitrile-2% glacial acetic acid (13:12:10, v/v), with a UV detector setting at 242 nm. After validation, the method was used for preliminary investigation of the pharmacokinetic profiles of dehydrotumulosic acid administrated in Ling-Gui-Zhu-Gan decoction.