A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed for the determination of nodakenin in rat plasma. The concentration of nodakenin was determined in plasma samples after deproteinization with methanol using hesperidin as internal standard. HPLC analysis was performed on a Diamonsil C18 analytical column using acetonitrile-water (25:75, v/v) as the mobile phase and a UV detection at 330 nm. This method was validated in terms of recovery, linearity, accuracy and precision (intra-and inter-day variation). The extraction recoveries were 91.3 ฯฎ 10, 87.8 ฯฎ 4.8 and 92.6 ฯฎ 5.1 at concentrations of 0.500, 5.00 and 40.0 mg/mL, respectively. The standard curve for nodakenin was linear (r 2 ี 0.99) over the concentration range 0.250-50.0 mg/mL with a lower limit of quantification of 0.250 mg/mL. The intraand inter-day precision (relative standard deviation, RSD) values were not higher than 12% and the accuracy (relative error, RE) was within ฯฎ5.8% at three quality control levels. The validated method was successfully applied for the evaluation of the pharmacokinetics of nodakenin in rats after oral administration of Rhizoma et Radix Notopterygii decoction and nodakenin solution.