Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. Next-generation proteomics, relying on ultra-rapid and subparts-per-million mass spectrometry analyzers, is able to offer in-depth insights into the molecular players sustaining the physiology of complex organisms. Identifying and quantitating thousands of proteins has become, over the past decade, a routine task for most proteomic platforms with the development of high-throughput shotgun proteomics. The interpretation of large-scale MS/MS data is only possible if a highquality database of nucleic acid sequences is available. Homology-driven proteomics using cross-species matching is a first alternative if the genome of interest is unknown, and de novo sequencing (i.e. interpretation of MS/MS data to establish the exact sequence of each peptide from scratch) is another possibility. However, major drawbacks of these two approaches lead to a scarcity of results as soon as a non-model organism, distantly related to a sequenced organism, is analyzed. Indeed, only highly conserved and ubiquitous proteins will be identified and carefully annotated with such approaches.