Vitexin is a natural flavonoid compound with multiple pharmacological activities and is extracted from the leaves and seeds of Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. However, the metabolite characterization of this component remains insufficient. To establish a rapid profiling and identification method for vitexin metabolites in rat urine, plasma and faeces after oral administration using a UHPLC-Q-Exactive orbitrap mass spectrometer were coupled with multiple data-mining methods. In this study a simple and rapid systematic strategy for the detection and identification of constituents was proposed based on UHPLC-Q-Exactive Orbitrap mass spectrometry in parallel reaction monitoring mode combining diagnostic fragment ion filtering techniques. A total of 49 metabolites were fully or partially characterized based on their accurate mass, characteristic fragment ions, retention times, corresponding ClogP values, and so on. It is obvious that C-glycosyl flavonoids often display an [M+H-120]+ ion that represents the loss of C4H8O4. As a result, these metabolites were presumed to be generated through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation, ring cleavage and their composite reactions. Moreover, the characteristic fragmentation pathways of flavonoids, chalcones and dihydrochalcones were summarized for the subsequent metabolite identification. The current study provided an overall metabolic profile of vitexin which will be of great help in predicting the in vivo pharmacokinetic profiles and understanding the action mechanism of this active ingredient.