Toad venom, a dried product of secretion from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, has had the therapeutic effects of hepatocellular carcinoma confirmed. Bufalin and cinobufagin were considered as the two most representative antitumor active components in toad venom. However, the underlying mechanisms of this antitumor effect have not been fully implemented, especially the changes in endogenous small molecules after treatment. Therefore, this study was designed to explore the intrinsic mechanism on hepatocellular carcinoma after the cotreatment of bufalin and cinobufagin based on untargeted tumor metabolomics. Ultraperformance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was performed to identify the absorbed components of toad venom in rat plasma. In vitro experiments were determined to evaluate the therapeutic effects of bufalin and cinobufagin and screen the optimal ratio between them. An in vivo HepG2 tumor-bearing nude mice model was established, and a series of pharmacodynamic indicators were determined, including the body weight of mice, tumor volume, tumor weight, and histopathological examination of tumor. Further, the entire m
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