<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Introduction</jats:title><jats:p>Radix Stemonae, first recorded in the herbal ‘<jats:italic>Additional Records of Famous Physicians</jats:italic>’ (Mingyi Bielu), has been widely used as an oral anti‐tussive agent in traditional Chinese medicine (TCM) for thousands of years in China. Current analysis methods are usually time‐consuming and inaccurate.</jats:sec><jats:sec><jats:title>Objective</jats:title><jats:p>The aim of this study was to develop a more rapid and sensitive method to detect the active compounds in Radix Stemonae.</jats:sec><jats:sec><jats:title>Methodology</jats:title><jats:p>A rapid, sensitive and selective ultra‐performance liquid chromatography–high‐resolution mass spectrometric (UPLC–HDMS) method with automated data analysis software MetaboLynx<jats:sup>TM</jats:sup> was developed for analysis and identification of the bioactive components and their metabolites in rat plasma following oral administration of Radix Stemonae extract.</jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Twenty‐nine compounds in the Radix Stemonae extract and 47 compounds in the rat plasma samples were detected in the MS<jats:sup>E</jats:sup> mode in a single injection within 21 min. Among the compounds detected, 23 were identified <jats:italic>in vitro</jats:italic> and 42 were identified in rat plasma, either by comparing the retention time and mass spectrometry data with those of reference compounds or by mass spectrometry analysis and retrieving the reference literature.</jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>It is concluded that the screening capability of MetaboLynx<jats:sup>TM</jats:sup>, together with the utilisation of MS<jats:sup>E</jats:sup> in structural elucidation, can facilitate a rapid and comprehensive detection and effective structural characterisation of <jats:italic>Stemona</jats:italic> alkaloids in Radix Stemonae extract, which could be applied to other Chinese medicine. The results provide helpful chemical information of Radix Stemonae extract for further pharmacology and active mechanism research. Copyright © 2012 John Wiley & Sons, Ltd.</jats:sec>