Mori Fructus (MF) is a famous edible fruit of Morus alba L. as well as traditional Chinese medicine (TCM). Moreover, exposure behavior of complex components in vivo is a necessary way to elucidate active substances in TCMs. However, the effective discovery of active ingredients from MF in vivo is still a challenge for researchers. In this study, an integrated strategy with chemical characterization, in vivo metabolism, chemical isolation, and activity evaluation was established and applied in targeted discovery of potential lipid-lowering substances in MF. First, an ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry method was established to characterize various chemical components in MF extract. Second, with the automatic matching (in-house database), discriminant ions analysis and metabolite software prediction, the metabolic profiling of different types of MF was elucidated in vivo. And the compounds from MF with high MS response in vivo were discovered. Third, according to LC-MS information of fractions, these compounds were isolated and identified by NMR. Finally, the isolated compounds were evaluated for the lipid-lowering activity on determination of triglyceride levels in high fructose-induced HepG2 cells at different concentrations. And PCSK9 inhibitory and LDL-R promoting activity were measured by western blot experiment. As a result, a total of 72 constituents were characterized in MF. After oral administration of MF extract, 16 prototypes and 33 metabolites were rapidly screened out. And the metabolism features of alkaloids, flavonols and organic acids were further revealed. Six alkaloids (morusimic acid A-D, G, F), with high MS response in vivo and no reference standards on the market, were isolated guided by LC-MS and identified by NMR. Among them, morusimic acid A, B, G and F could reduce triglyceride levels in fructose-induced HepG2 cells. Moreover, with western blot experiment, morusimic acid A, B, G and F could inhibit the expression of PCSK9 protein. And morusimic acid A, B and F could increase the expression of LDL-R protein. This work provides meaningful information for the discovery of potential compounds in MF for the treatment of obesity and hyperlipidemia, along with a new approach for exploring effective compounds from complex systems. © 2023 The Author(s)