Abstract: This study aims to rapidly screen and identify pancreatic lipase (PL) and cholesterol esterase (CE) inhibitory peptides from hemp seed protein hydrolysates (HSPHs), and validate their activities, accompanied by preliminarily analyzing their molecular mechanisms. Initially, the PL and CE inhibitory activities of HSPHs were explored under different buffer systems. Subsequently, a combination of ultrafiltration, peptidomics, and virtual screening methods was employed for the rapid identification and screening of PL and CE inhibitory peptides. Finally, their activities were validated, and molecular docking was used to investigate the types of molecular interactions involved. The results indicated that, compared to conventional buffer systems, the simulated intestinal fluid system reduced the inhibitory effect of HSPHs on PL but enhanced their inhibitory effect on CE. Therefore, subsequent tests of PL and CE inhibitory activities were conducted using the simulated intestinal fluid system. The fractions of HSPHs smaller than 3 kDa, released by alcalase and papain, exhibited the highest PL/CE inhibitory activities. After identifying the peptide sequences, 136 peptides with higher content and potential activity were selected to establish a candidate peptide library. From this library, 14 peptides with lower binding energies were identified as PL and CE inhibitory peptides. In vitro activity validation demonstrated that APAM and RLPA exhibited significant inhibitory effects on both PL and CE. Molecular docking results suggested that APAM and RLPA might inhibit the activities of PL and CE by forming hydrogen bonds and hydrophobic interactions with the residues at the enzyme active sites and surface loops, thereby restricting the exposure of the active sites and the entry of substrates. This study indicates that hemp seeds are a potential source for developing lipid digestion enzyme inhibitory peptides, providing a reference for the subsequent development of lipid-lowering peptide products from hemp seeds.