Mulberry 1-deoxynojirimycin (DNJ, a potent α-glycosidase inhibitor) has been investigated thoroughly for its analytical methods and therapeutic potential against diabetes, whereas little attention has been given to other iminosugars such as 2-O-α-d-galactopyranosyl-DNJ (GAL-DNJ) and fagomine. For instance, concentration and composition of these iminosugars in mulberry leaves as well as sericulture products have not been fully characterized due to lack of suitable analytical methods. Here we developed a simultaneous determination method for DNJ, GAL-DNJ, and fagomine using hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS). When mulberry leaf extracts were subjected to HILIC-MS/MS with multiple reaction monitoring (MRM), individual iminosugars could be separated and detected. The developed method is sufficiently sensitive for determining iminosugars in mulberry leaves as well as silkworms, providing new information (e.g., different amounts of iminosugars in mulberry leaf varieties; high DNJ and low GAL-DNJ in the silkworm body, especially in the blood) that is useful for producing iminosugar-rich products for nutraceutical purposes.