The characteristic fragmentation of four strychnos alkaloid compounds has been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). In the obtained product ions spectra, some diagnostic neutral losses were observed, which are very helpful for the identification of strychnos alkaloids. According to the structural information collected from spectrum, four strychnos alkaloids have been identified on-line without time-consuming isolation, and two unknown alkaloids have been detected in the extract of crude alkaloids of Strychnos nux-vomica L. An effective and rapid HPLC-ESI-MSn method for analyzing constituents in Strychnos nux-vomica L. has been established and proved. © 2014 Copyright Taylor and Francis Group, LLC.