In general, plant contains a certain degree of resistance against insect pest and digestive enzymes inhibitors could be produced for plant defense. A serine-proteinase inhibitor from timbauba, which inhibited trypsin, chymotrypsin and plasma Kallikrein and an additional inhibitor with activity directed against papain and bromelain have been related (Oliva et al., 1992). Here, we report an inhibitory activity of timbaúba proteins against serine proteinases from A. grandis (cotton boll weevil) and S. fugiperda (fall armyworm) larvae. The assays were done using protein seeds fraction (0-60% ammonium sulphate) according Kunitz (1947) and the results shown 16% and 45% of inhibition against the two insects pest, pespectively. In addition, the amylase inhibitory activity was tested against the same insects, according to Bernfeld (1955). Here, we observed 17% and 65% of inhibition, respectively. Thus, the amylase inhibitor has been purified through chromatography. Protein fraction was applied in an affinity chromatography column, Red-Sepharose Cl-6B and the retained peak was applied in HPLC (Surphedex 75 10/30 column). By SDS-PAGE were visualized three majority protein bands in retained peak from Red-Shepharose. The molecular masses were determined by MALDI TOF as 12,858, 13,473 and 16,503 Da. After final purification, this inhibitor will be used in biological tests against S. fugiperda larvae and could represent a good candidate for the production of transgenic plants resistant to insect pest mentioned above.