Despite the importance of invertebrates in aquatic systems, we lack specific biomarkers to assess the effects of endocrine disruption (ED) on these animals. This can be attributed to a poor knowledge of their endocrine systems and to the absence of genomic data for non-model species. The development of relevant tools for hazard assessment of ED is currently a challenge for crustacean species used in aquatic ecotoxicology. Our research teams have conducted a “proteogenomic” approach to generate a large transcriptomic and proteomic dataset for the amphipod crustacean Gammarus fossarum, a sentinel species used widely in aquatic ecotoxicology in Europe. However, among the 2257 MS-certified proteins obtained with this approach, key proteins involved in hormonal regulation that could be potential candidates for endocrine disruption biomarkers were not identified. Therefore, the aim of this study was to identify, in G. fossarum, some key players involved in the endocrine regulation of crustaceans/amphipods in order to propose candidate biomarkers of endocrine disruptions in this species. For this, we first established a list of candidate genes known to play an essential role in hormonal systems of crustaceans and insects: nuclear hormone receptors, other regulatory genes, enzymes of the hormone metabolism and hormone-regulated genes. Using sequence similarity and phylogenetic analyses, we identified similar sequences of three candidate genes in our transcriptomic G. fossarum database: the nuclear receptors RXR and E75, and the regulator broad-complex. The three genes were cloned in order to obtain reliable nucleotide sequences and primers for the subsequent expression studies. Their functional validation and relevance as biomarkers was then performed by studying gene expression during the female reproductive cycle, and after laboratory exposure to model ED chemical compounds (pyriproxyfen and tebufenozide).