Fluorescein diacetate-labeled HepG2 cells model and flouresence automatic microscopy screening assay were used for fast screening 23 components from Toosendan Fructus, in which 5 components showed significant toxicity on HepG2 cells. The 10 compounds in the 2 components were tentatively identified with LC-MS(n), and 3 of them (meliasenin B, trichilinin D and 1-O-tigloy-1-O-debenzoylohchinal) were prepared and identified. Further experiments showed that the 3 compounds displayed dose-dependent toxicity on HepG2 cells, suggesting that these compounds in Toosendan Fructus may cause hepatotoxicity.