Abstract: Harmine is one of β-carboline alkaloid that significantly inhibits the multivulva phenotype of let-60(n1046gf), a worm called Caenorhabditis elegans. Therefore, harmine has an important potential application in pancreatic cancer. Metabolic research in vivo is one of the important ways of pharmacodynamic substance research, which can illuminate the existent form medicine in vivo and confirm the real active constituent of medicine. The fragmentation pathway of harmine was analyzed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS), and the possible fragmentation pathways and characteristic fragment ions were determined. The metabolites of harmane in Caenorhabditis elegans were studied by UPLC-Q-TOF MS. The MRM data acquisition model was used to study the content of harmine in the nematode worms at different time after administration by the semi-quantitative analysis. The results show that the fragment pathways of harmine are mainly RDA cleavage, including the loss of methyl groups and hydroxyl groups, and the cracking of indole ring. The metabolites of demethylated (m/z 199) and hydroxylated (m/z 229) of harmine (m/z 213) were detected in positive ion mode. The results of semi-quantitative study show that the metabolism of nematodes is strong in 0.5 h, and the content of harmine is decreased by about 80%, the metabolism rate slows down after 5 h. The study can lay the foundation for the development of new drugs for the treatment of pancreatic cancer.