Trypsin inhibitors and chymotrypsin inhibitors are protease inhibitors of protein nature. Occurring naturally in legumes, cereals, and some other species, they are considered key antinutritional factors, since they can interfere with protein digestions, induce hypersecretion of trypsin and chymotrypsin in animal digestive tracts, and lead to pancreatic enlargement and growth suppression of animals. Paradoxically, during the past few decades, some protease inhibitors have been found to be beneficial in humans and animals by exerting therapeutic effects. Therefore, protease inhibitor activities remaining in food and feed products have been considered important parameters, due to their significant effects on animal nutrition and human health. Historically, trypsin inhibitor activity has been a priority parameter for measurement, but lately determination of chymotrypsin inhibitor activity has also gained attention. This chapter covers the latest method for measuring trypsin inhibitor activity and the latest method for measuring chymotrypsin inhibitory activity, after providing a brief overview of the method development over the past half century for measuring activities of the protease inhibitors. The latest methods feature improved sensitivity and accuracy and can express inhibitory activities in both arbitrary units and absolute amounts of trypsin or chymotrypsin inhibited for easy comparison among studies. A detailed description of the latest method for determining chymotrypsin inhibitor activity is also provided.