Trypsin is a key protease related to digestion and absorption of proteins, which its inhibition must be studied when natural compounds, such as flavonoids, are used as part of alternative treatments for obesity and diabetes mellitus type 2, since trypsin and together with other pancreatic enzymes worked at small intestine. Considering that flavonoids are good lipase and amylase inhibitors, trypsin-flavonoids interactions were analyzed through UV-Vis, intrinsic and extrinsic fluorescence spectroscopies, circular dichroism, and molecular docking. The interaction between porcine pancreas trypsin and five flavonoids: hesperetin (HES), luteolin (LUT), quercetin (QUE), catechin (CAT), and rutin (RUT) was evaluated. Most of them exhibited a mixed-type mode. LUT was the best trypsin inhibitor (e.g., lower IC50, 45.20±1.00 µM). All flavonoids-trypsin complexes showed static quenching, and QUE and LUT exhibited higher affinity (higher Ka values, 0.90±0.10 and 1.60±0.20·10-1 mM-1, respectively). Hydrophobic interactions between trypsin and flavonoids were predominant.