BACKGROUND: Cnidii Fructus (CF) is a herbal medicine with pharmacological activities such as antitumor, antiviral, antiallergic, antipruritic effects, and so on. OBJECTIVE: In this study, an ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC- MS/MS) method was prepared and verified to measure the concentrations of seven analytes (bergapten, xanthotoxol, xanthotoxin, imperatorin, osthole, isopimpinellin, isoimperatorin) in HepG2 cells. METHODS: The separation of seven analytes was performed on an ACQUITY UPLC® BEH C18 column (2.1×100 mm, 1.7 μm) with a gradient mobile phase system of 0.1% formic acid/water and acetonitrile. RESULTS: The CV of analytes was within 7.77%, and the bias was in the range of -5.43%-3.84%. The matrix effects of analytes ranged from 92.95% to 104.58%, and the extraction recoveries ranged from 76.45% to 104.69%. The relative standard deviation of stability results was less than 8.21%, indicating that seven analytes were stable. CONCLUSION: The method was successfully applied to the determination of the content of seven analytes of CF extracts by UPLC-MS/MS, and the results will provide a reference for the cellular pharmacokinetics of CF.