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Insects can recognize invading pathogens and initiate an immune response. Among them, Drosophila has emerged as an invertebrate model for investigating innate immune responses in which antimicrobial peptides play a crucial role. In the present study, immune-induced antimicrobial peptides were characterized in D. melanogaster and D. ananassae using the agar well diffusion method, HPLC, SDS-PAGE and LC-MS/MS after infection with either S. aureus or E. coli. The HPLC revealed two and three differentially induced components, respectively, in D. melanogaster and D. ananassae flies infected with S. aureus and E. coli. The tricine SDS-PAGE analysis also revealed two and five differentially induced proteins, respectively, in D. melanogaster and D. ananassae infected with E. coli. In E. coli infected flies, the ~6 kDa band was produced at higher level. Based on LCMS/MS and Mascot analysis, the peptide was identified as a putative cecropin A-like peptide, and the data suggested that both species of Drosophila have exhibited a clear immune response. The flies were also able to discriminate between bacteria, as this putative cecropin A-like peptide was produced in flies infected with E. coli but not S. aureus.