Serine proteases (SPs) and serine protease homologs (SPHs) play essential roles in insect physiological processes including digestion, defense and development. Studies of insect genomes, transcriptomes and proteomes have generated a vast amount of information on these proteins, dwarfing the biological data acquired from a few model species. The large number and high diversity of homologous sequences makes it a challenge to use the limited functional information for making predictions across a broad taxonomic group of insects. In this work, we have extensively updated the framework of knowledge on the SP-related proteins in Drosophila melanogaster by identifying 52 new SPs/SPHs, classifying the 257 proteins into four groups (CLIP, gut, single- and multi-domain SPs/SPHs), and detecting inherent connections among phylogenetic relationships, genomic locations and expression profiles for 99 of the genes. Information on the existence of specific proteins in eggs, larvae, pupae and adults is presented to facilitate future research. More importantly, we have developed an approach to reveal close homologous or orthologous relationships among SPs/SPHs from D. melanogaster, Anopheles gambiae, Apis mellifera, Manduca sexta, and Tribolium castaneum thus inspiring functional studies in these and other holometabolous insects. This approach is useful for tackling similar problems on large and diverse protein families in other groups of organisms.