ObjectiveTo identify the chemical components in traditional Chinese herbal medicine Veratrum nigrum L. using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). MethodsAn Agilent Eclipse Plus C18(2.1 mm×100 mm,1.8 μm)was used for isolation and identification of the chemical components in Veratrum nigrum L., with a mobile phase using 0.1% formic acid aqueous solvent (A) and ACN (B) for gradient elution. The data were collected by the positive ion mode using ESI ion source. ResultsThirty-two chemical compounds were identified from Veratrum nigrum L.. The chemical structures of three pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. ConclusionChromatographic characters of 32 chemical compounds in Veratrum nigrum L. can be achieved in single run by UHPLC-Q-TOF/MS, which paves a way for further studying the metabolism and action mechanism of Veratrum nigrum L.