Using an in-depth Mass Spectrometry based proteomics approach, we provide a comprehensive characterization of the hemolymphatic proteome of adult flies upon bacterial infection. We detected and quantified changes in abundance of several known immune regulators and effectors, including multiple antimicrobial peptides, peptidoglycan-binding proteins and serine proteases. Comparison to previously published transcriptomic analyses reveals a partial overlap with our dataset, indicating that many proteins released into the hemolymph upon infection may not be regulated at the transcript level. Among them, we identify a set of muscle-derived proteins released into the hemolymph upon infection. Finally, our analysis reveals that infection induces major changes in the abundance of proteins associated with mitochondrial respiration. This study uncovers a large number of previously undescribed proteins potentially involved in the immune response.