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Summary: Currently, the identification of herb metabolites is challenging due to a lack of clear standards. Here, using Gelsemium as an example, we present a protocol for characterizing target components of herbs. This approach utilizes high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry guided by an in-house herb metabolite database based on reported studies and mass spectrometry. We describe steps for creating an in-house database, preparing and detecting samples, processing data, and characterizing compounds. This approach offers a reference for future research on the identification of herb metabolites.For complete details on the use and execution of this protocol, please refer to Liu et al. (2017).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.