Introduction For centuries, Semen Strychni (the ripened seeds of Strychnos nux-vomica) has been used extensively as a herbal medicine in Asian countries. However, the chemical composition of the dihydroindole-type alkaloids contained in processed Semen Strychni is not fully understood. Objective To develop an improved strategy using mass defect filtering (MDF) in combination with MS n analysis and theoretical calculations for identification and structural characterisation of dihydroindole-type alkaloids in processed Semen Strychni extracts. Methods The experimental work was conducted using a high-performance liquid chromatography coupled with electrospray ionisation ion trap time-of-flight mass spectrometry (HPLC-ESI/IT-TOF/MS) system. Upon acquisition of full-scan MS data, the potential dihydroindole-type alkaloids were screened using a well-defined mass defect range of 50 mDa. With the assistance of MSn analysis, the diagnostic fragment ions (DFIs) were used as primary screening references for targeting the characteristic analogues. For better discrimination of the isomers, quantum chemical calculations were utilised to provide additional structural information. Results Twenty-four dihydroindole-type alkaloids, including four that were previously not described, were tentatively identified. Conclusion A new, rapid and sensitive method was developed for the discovery and characterisation of dihydroindole-type alkaloids in extracts of processed Semen Strychni. The successful application of this method indicates a potential for adaptation to other classes of natural product from other sources. Copyright © 2013 John Wiley & Sons, Ltd.