This paper describes a simple, rapid, and validated reversed-phase high-performance liquid chromatographic method developed for the determination of 4 major bioactive constituents, namely, chlorogenic acid, ferulic acid, senkyunolide A, and Z-ligustilide in Rhizoma Cnidii extract. A Capcell Pak C18 chromatographic column (150 x 4.6 mm, 3 microm) was used with mobile phases consisting of 0.1% formic acid, acetonitrile, and methanol at a flow rate of 0.8 mL/min and UV detection at 285 nm. Comprehensive validation of the method included evaluation of linearity, repeatability, recovery, and stability. Excellent linear behavior (r2>0.99) was observed over the concentration range of 2-100 microg/mL for the compounds under investigation. Repeatability and accuracy were evaluated by intra- and interday assays; the relative standard deviation (RSD) values were < or = 5.37% and accuracies ranged from 97.1 to 104.9%. Recoveries of the compounds ranged from 94.2 to 104.2% with RSD values of < or = 9.50%. The developed method was successfully applied to the analysis of ethanolic extracts of Rhizoma Cnidii samples. As a result, the concentrations of chlorogenic acid, ferulic acid, Z-ligustilide, and senkyunolide A were determined to be 0.84-5.35, 0.45-1.65, 0.74-4.39, and 0.32-1.14 mg/g herb, respectively. Thus, the developed method was found to be accurate and reproducible and is considered suitable for the qualitative and quantitative analysis of Rhizoma Cnidii for bioactive compounds.