Abstract: A high-performance liquid chromatography (HPLC) analytical method was established for the determination of xanthotoxin, bergapten, and osthole in Cnidii Fructus formula granule, a traditional Chinese medicine. The HPLC determination was performed using Welch Ultimate Plus C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile-water and gradient elution. The flow rate was set at 1.2 mL/min, the detection wavelength was 248 nm, the column temperature was maintained at 30 ℃, and the injection volume was 12 μL. By improving the mobile phase composition and the gradient elution program, with the use of a common particle size C18 column, the three components, namely xanthotoxin, bergapten, and osthole in Cnidii Fructus formula granule, could achieve baseline separation from the adjacent peaks within a relatively short time (less than 30 min). The results of the methodological evaluation indicated that the established method was accurate and reliable, and the experimental conditions were simple and convenient. This method was also applied to the analysis of actual samples of the formula granules of Cnidii Fructus.