A sensitive and specific method was proposed and validated for the simultaneous determination of 17 poisonous alkaloids in human blood and gastric juice by liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS=MS). After mixing with boric acid buffer solution (pH 9), the samples were extracted with chloroform followed by chloroform-ether (2:1, v=v). The simultaneous qualitative and quantitative analysis of 17 alkaloids, including koumine, atropine, scopolamine, brucine, strychnine, aconitine, bulleyaconitine A, ephedrine, pilocarpine, lobeline, ergometrine, anabasine, oxymatrine, theophylline, colchicine, tetrahydropalmatine, and eserine, were performed by using ESI þ in multiple-reaction monitoring (MRM) mode. Under the optimized conditions, the calibration curves of the 17 compounds presented good linearity (r=0.9952) in the ranges of 0.5-50 lg=L and 0.5-500 lg=L. The quantitation limits (LOQs) and detection limits (LODs) ranged from 0.5-1 and 0.1-0.5 lg=L, respectively. The average extraction recoveries were obtained in the range of 81.7-102.3 %. The intra-day and inter-day relative standard deviations (RSDs) were less than 10% and 15 %, respectively. The results indicated that the method was simple, rapid, selective, sensitive, and stable enough to examine toxic alkaloids and was suitable for poisoning diagnosis and forensic toxicology.