Simple SummaryAntibiotic resistance is a worldwide social and health crisis. The search for therapeutic alternatives, including the use of antimicrobial peptides (AMPs), is critical. AMPs are small molecules synthesized by a wide range of living organisms. Microbiological and mass spectrometric techniques were used to examine peptides in the hemolymph of larvae of the scavenger insectHermetia illucens(Diptera, Stratiomyidae) after infection withEscherichia coliorMicrococcus flavus, as well as uninfected larvae, used as control. Microbiological assays allowed us to confirm antimicrobial activity ofH. illucensAMPs, while via mass spectrometry we identified a set of 33 AMPs, expressed in different conditions: 20 AMPs were expressed in all the analyzed conditions, while 13 were differentially expressed after Gram negative or Gram positive bacterial challenge. Differentially expressed AMPs may be responsible for a more specialized action.AbstractAntimicrobial peptides (AMPs) are a chemically and structurally heterogeneous family of molecules produced by a large variety of living organisms, whose expression is predominant in the sites most exposed to microbial invasion. One of the richest natural sources of AMPs is insects which, over the course of their very long evolutionary history, have adapted to numerous and different habitats by developing a powerful innate immune system that has allowed them to survive but also to assert themselves in the new environment. Recently, due to the increase in antibiotic-resistant bacterial strains, interest in AMPs has risen. In this work, we detected AMPs in the hemolymph ofHermetia illucens(Diptera, Stratiomyidae) larvae, following infection withEscherichia coli(Gram negative) orMicrococcus flavus(Gram positive) and from uninfected larvae. Peptide component, isolated via organic solvent precipitation, was analyzed by microbiological techniques. Subsequent mass spectrometry analysis allowed us to specifically identify peptides expressed in basal condition and peptides differentially expressed after bacterial challenge. We identified 33 AMPs in all the analyzed samples, of which 13 are specifically stimulated by Gram negative and/or Gram positive bacterial challenge. AMPs mostly expressed after bacterial challenge could be responsible for a more specific activity.