A liquid chromatography-electrospray ionization-ion trap mass spectrometric（LC-ESI-ITMS） method was develop for the simultaneous analysis of three common analeptics and their in-vitro metabolites.Analeptics were incubated individually with mouse liver microsome S9 fraction firstly，and then the incubation samples were pooled together and analyzed by LC-ESI-ITMS in full-scan mode.Analeptics and their metabolites were identified from the determination of molecular ions.The metabolites detection were confirmed from tandem mass spectrometric（MSn） analysis.With the advanteges of rapidness，high accuracy，good selectivity and sensitivity，the method was suitable for the analysis of three common analeptics and their in-vitro metabolites.