A rapid and sensitive high-performance liquid chromatography-electrospray mass spectrometric method has been developed for the determination of betaine in Lycium chinense fruits. Betaine was analyzed on a system consisting of a NH2 stationary phase and a mobile phase of water-acetonitrile (25:75) by isocratic elution for 40 min. Betaine was identified and quantitated by electrospray ionization mass spectrometry with selected ion monitoring of the protonated ion [Betaine+H]+ and clustered ions [nBetaines+H]+. The limit of detection for betaine by this method was ca. 0.2 ng/ml and the relative standard deviations of the assay (intra- and inter-day) were less than 8.1%.