Background: Fructus Corni (FC), a well-known traditional Chinese medicine (TCM), derived from the dry ripe sarcocarp of Cornus offi cinalis Sieb. et Zucc (Cornaceae), has been widely prescribed to treat disease in China for centuries. It has attracted increasingly much attention as one of the most popular and valuable herbal medicine in clinic. However, the systematic analysis of the chemical constituents of FC is diffi cult to determine and remain unclear. Materials and Methods: In this work, a rapid, sensitive, and reliable ultra-performance liquid chromatography-electrospray ionization/ quadrupole-time-of-fl ight high-defi nition mass spectrometry (UPLC-ESI/QTOF/MS) with automated data analysis (MetaboLynx TM ) in negative ion mode were established to characterize the chemical constituents of FC and simultaneously identify components in blood after oral administration of FC, respectively. The analysis was performed on a Waters UPLC TM HSS T 3 (2.1 × 100 mm, 1.8 μm) using gradient elution system. MS/MS fragmentation behaviors were proposed for aiding the structural identifi cation of the components. Results: With optimized conditions, a total of 34 peaks were obtained from FC, 23 of which were tentatively characterized by comparing the retention time and mass spectrometry data and retrieving the reference literatures. Of note, the 25 compounds were identifi ed after oral administration of FC, which might be the potential active components in vivo. Conclusion: The present study demonstrates the potential of UPLC-ESI/QTOF/MS approach for the rapid and reliable characterization of the metabolites of natural products.