A high-performance liquid chromatographic method was developed for the determination of macelignan in rat plasma and applied to the pharmacokinetic study of macelignan in rats. Chromatographic separation was achieved on a conventional ODS column with the mobile phase of water: acetonitrile: methanol = 35:32.5:32.5 (v/v/v %). The flow rate of isocratic elution was 1 mL/min and peaks were detected at 240 nm. The limit of detection was 10 ng/mL and the limit of quantitation was 20 ng/mL. The calibration curve was linear over the concentration range of 50-5000 ng/mL. Intra-and inter-day precision for the assay over the concentration range was below 10 % and the accuracy ranged between 96.0-107% for intra-day and 98.8-114% for inter-day, respectively. The method was applied to the single dose pharmacokinetic study of macelignan in rats and the results showed that this HPLC method was adequate to support the in vivo pharmacokinetic study of macelignan.