•Simultaneous determination of DNJ, fagomine and DAB in rat plasma was done.•A simple, selective and sensitive LC–MS/MS method was developed and validated.•The method was applied to a pharmacokinetic study of mulberry twig in rats.Fagomine, 1-deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) are the major bioactive constituents in the active fraction of alkaloids from the traditional Chinese medicine mulberry twig (Ramulus Mori, Chinese name Sang Zhi), which has a strong activity on α-glucosidase in vitro and in vivo. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of DNJ, fagomine and DAB in rat plasma. Plasma samples were prepared using a simple protein precipitation by the addition of 1% volume of Tris and two volumes of methanol–acetonitrile. The analytes and internal standard (IS, miglitol) were chromatographed in an XBridge™ amide column with a gradient mobile phase of acetonitrile–water (0.1% ammonium hydroxide) at a flow rate of 0.7 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source in positive ion mode by multiple reaction monitoring (MRM) mode. Linear detection responses were obtained for DNJ ranging from 5.00 to 5000.00 ng/mL, 10.00 to 2500.00 ng/mL for fagomine and DAB. The lower limits of quantification (LLOQs) were 5.00, 10.00, 10.00 ng/mL for DNJ, fagomine and DAB, respectively. Intra-day and inter-day precisions (R.S.D.%) were within 10% for three analytes with accuracies (R.E.%) less than 12%. The mean recoveries of analytes were greater than 85%. All analytes were proved to be stable during the sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the three alkaloids in rats after oral administration of the active fraction of alkaloids from mulberry twig.