Plant derived α-amylase inhibitors are proteinaceous molecules that regulate the enzyme activity in plants and also protect plants from insect attack. In the current study, 28 accessions of 19 plant species were screened for their α-amylase inhibitory activity. The durum wheat varieties, Beni Suef-1 and Beni Suef-5, showed strong α-amylase inhibitory activity and were subjected to further purification studies using ammonium sulfate fractionation and DEAE-Sephadex G-25 column. The isolated inhibitors were found to be stable at temperatures below 80°C with maximum activity obtained at 40−50°C. Also, they were stable in a wide pH range (2−12). The ion exchange products of purified α-amylase inhibitors from Beni Suef-1 and Beni Suef-5 varieties showed a molecular weight of 16 and 24 kDa, respectively. The purified α-amylase inhibitors were tested against Tribolium castaneum and Callosobruchus maculatus both in vitro and in vivo. There was linear inhibition of α-amylase activity with increasing inhibitor concentration until saturation was reached. Beni Suef-5 α-amylase inhibitor was more potent against α-amylase with lower IC50 values than Beni Suef-1 α-amylase inhibitor except in the case of T. castaneum larva. Kinetics analysis revealed that Beni Suef-1 and Beni Suef-5 α-amylase inhibitors are non- -competitive types of inhibitors with high affinity toward α-amylase of T. castaneum and C. maculatus. Results of the in vivo studies demonstrated that α-amylase inhibitors isolated from durum wheat, Beni Suef-1 and Beni Suef-5 varieties, were very effective in inhibiting the development of T. castaneum and C. maculatus and could be used for future studies in developing insect resistant transgenic plants approaching α-amylase inhibitor genes.