Polyhydroxylated alkaloids, or alkaloidal iminosugars, which exhibit potent bioactivity as glycosidase inhibitors, can be used as potential therapeutic agents or as tools for probing into biological recognition processes. Mulberry has aroused considerable research interest in recent decades as it is rich in polyhydroxylated alkaloids, in particular it is a major natural source of 1-deoxyjirimycin (DNJ). In the present work, we achieved the rapid and direct detection of polyhydroxylated alkaloids in mulberry using leaf spray mass spectrometry. By semi-quantitative analysis, the relative content of DNJ in different plant materials from mulberry trees was recorded and the experimental results provided us an insight into the distribution of DNJ in mulberry, which can be instructive and informative for the practical production of functional or medical mulberry food. In addition, thanks to its fast, simple and high throughput characteristics, the leaf spray method exhibits promising application in the rapid screening of lead compounds from natural plants during drug development.