A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C 18 CapcellACR analytical column (150mm×4.6mm I.D. 5μm) with a mobile phase consisting of acetonitrile–0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330nm. The lower limit of detection and quantification were 9 and 29.2ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2–18250ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.