Identifying new genes involved in virulence and drug resistance may hold the key to a better understanding of parasitic diseases. The proteogenomic profiling of various Leishmania species, the causative agents of leishmaniasis, has identified several novel genes, N- and C-terminal extensions of proteins, and corrections of existing gene models. Various virulence factors (VFs) responsible for leishmaniasis have been previously annotated through a proteogenomic approach, including the C-terminal extension of heat shock protein 70 (HSP70). Furthermore, the diversity of VFs across Leishmania donovani, L. infantum, L. major, and L. mexicana was determined using phylogenetic analysis. Moreover, protein–protein interaction networks (PPINs) of VFs with HSPs aid in making significant biological interpretations. Overall, an integrated omics approach involving proteogenomics was used to identify and study the relationship among VFs with other interacting proteins, including HSPs. This chapter provides a step-by-step guide to the identification of new genes in Leishmania using a proteogenomic approach and their functional assignment using a bioinformatics-based approach.