Objectives: As a traditional Chinese medicine, lotus leaf was reported to have significant hepatoprotective effect. To explore the hepatoprotective mechanism of lotus leaf, a rapid and reliable UPLC-MS/MS method was conducted to simultaneously determine six specific endogenous substances including 5-oxoproline, phenylalanine, tryptophan, C18-phytosphingosine, lysophosphatidylcholine (16 : 0) and lysophosphatidylcholine (18 : 1). Methods: With the help of HPLC–FT-ICR-MS, the chemical constituents of louts leaf extract were elucidated. By observing histopathological changes and determining hepatotoxicity-related biochemical indicators, rat model of liver injury was developed and the hepatoprotective effect of lotus leaf was verified. With the developed UPLC-MS/MS method, six endogenous metabolites related to hepatotoxicity were monitored to investigate the hepatoprotective mechanism of lotus leaf. Key findings: In the qualitative analysis, a total of twenty compounds including ten flavonoids, nine alkaloids and one proanthocyanidin were identified. Based on the results of determining six endogenous metabolites related to hepatotoxicity, it was predicted that the hepatoprotective mechanism of lotus leaf might be related to glutathione metabolism, phenylalanine metabolism, tryptophan metabolism, sphingolipid metabolism and phospholipid metabolism. Conclusions: This study could be a meaningful investigation to provide mechanistic insights into the hepatoprotective effect of lotus leaf and further lay a theoretical basis for the clinical application of lotus leaf. © 2020 Royal Pharmaceutical Society