Chymotrypsin-like enzymes are involved in many physiological processes of insects including digestion, development, survival, and immunity. The enzyme cleaves the peptide bonds on C-terminal of the aromatic amino acids in proteins and release active peptides as well as amino acids required for growth, reproduction, and development of the insect. Due to the importance of food proteins on insect survival and development, more fundamental entomologists are researching the structures, functions, mechanisms, and interactions of the involved digestive enzymes. Discovering biochemical and structural properties of insect proteases can help develop rational control strategy based on specific protease inhibitors. Accordingly, the present study focuses on protein structural analysis, protein sequence alignment, phylogenetic analysis as well as conserved motif assessment from various insect species using different bioinformatics tools. The multiple sequence alignment revealed different conserved stretches of amino acids along with highly conserved catalytic sites (His, Asp, Ser) and ten conserved motifs were also discovered by MEME and MAST tools. The phylogenetic data suggest that the insect chymotrypsins might share a common ancestor. The three-dimensional structures of chymotrypsins were generated by I-TASSER, and the 3D model was further verified using PROCHECK, ERRAT, and Verify-3D. The protein – protein interactions network constructed by STRING 11 provided ten enriched pathways in Aedes aegypti (L.) chymotrypsin. Totally, the present work can provide new insights for designing alternative pesticides based on specific digestive enzyme inhibitors.