Introduction - Plants in the genus Stephania can produce diverse bioactive alkaloids. Stephania hainanensis is a medicinal plant that contains effective alkaloids. However, only 10 alkaloids have been reported in this species.Objective - To characterise the alkaloids in Stephania hainanensis using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS).Methods - An LC-QTOF-MS/MS method was developed for structural characterisation of the alkaloids in Stephania hainanensis. The chromatographic separation was performed on a phenyl column with gradient elution, and the tandem mass spectra were obtained by using an electrospray ionisation (ESI) interface in positive ionisation mode. Compound identification was based on the exact masses, fragmentation pathways, retention behaviours and related botanical biogenesis.Results - A total of 37 tetrahydroprotoberberine-, quaternary protoberberine-, aporphine-, proaporphine-, benzylisoquinoline- or bisbenzylisoquinoline-type alkaloids were identified or tentatively identified in a single LC run. Twenty-seven of these alkaloids, including the benzylisoquinoline-type of alkaloids, have not been previously reported in Stephania hainanensis. The possible fragmentation pathways of different types of alkaloids were proposed. Besides the general fragmentations, the characteristic losses of CH3N=CH2 were observed for the benzylisoquinoline and aporphine alkaloids with two methyl groups on the nitrogen.Conclusion - The LC-QTOF-MS/MS method enabled profiling and rational, but tentative, identification of diverse alkaloids in Stephania hainanensis. The results obtained may be helpful for understanding the bioactivity of S. hainanensis and evaluating the quality of this plant. Copyright (C) 2016 John Wiley & Sons, Ltd.