Abstract: An high-performance liquid chromatographic electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed for the identification of 6 alkaloids in food. A mixture of acetonitrile (A) and pH5 4mmol/L ammonium acetate aqueous solution (B) were used as the mobile phase for the chro-matographic separation on ZORBAX SB-C8 (2.1mm×150mm, 3.5μm) column in the gradient elution mode. Under positive electrospray ionization mode, the protonated molecularM+H+ions for betaine (m/z 118.2) , choline (m/z 104.2) , matrine (m/z 249.5) , atropine (m/z 290.3) , brucine (m/z 395.4) and aconitine (m/z 646.4) and retain time were obtained as qualitative analysis. The experiment results indicated that RSD of the precision was less than 0.59% for adding 1.0mg/mL of spike standard. This method was used in the actual sample Herba Cistanche, Rhizoma Dioscoreae, Lycium bar-barum L testing and the average extraction recoveries were in the range of 81.7%¹12.9%, 80.8%¹17.5%, 96.9%¹19.3%. A simple, rapid and accurate identification of 6 alkaloids was successfully achieved using the method.