We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila , regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5–11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.