The use of plants as a way to prevent and treat disease comes from ancient times. With the increasing return on consumption of plants for medicinal purposes, the plant-based medicines have gained greater appreciation. Protease inhibitors are compounds that can decrease activity of an enzyme. They are found in plants, especially in Fabaceae, Poaceae, and Solanaceae families, and show satisfactory use to treat diseases due to their regulatory activities and selective proteolytic degradation of target substrates. This study aimed to obtain protease inhibitors in the seeds of Punica granatum, due its use in traditional medicine. The alcohol-acid solution was used to extract and isolate the protease inhibitors in P. granatum seeds and Glycine max grains. The extract of P. granatum seeds showed similar concentration of protease inhibitors when compared to G. max. A 14 kDa band was detected by electrophoresis on polyacrylamide gel, which was characterized by high-performance liquid chromatography as protease inhibitors. The extract of P. granatum seeds showed a potent trypsin inhibitor activity (EC50 18.2 ng mL-1). This study showed for the first time the extraction, purification, and identification of protease inhibitors in Punica granatum seeds.