Biomarkers related to exposure to chemical compounds can be monitored to predict possible hazard on health of sentinel organisms. The routine use of these tools in biomonitoring faces several drawbacks, especially in invertebrates: the lack of robust species-specific quantification methods and the use of numerous biomarker-specific protocols that lead to very expensive biomonitoring strategies in time, cost and biological samples. Recently, “proteogenomics” emerged as a relevant strategy for the discovery of proteins in non-model organisms. With the combination of high throughput genomic and proteomic methodologies, Trapp et al. built a protein sequence database consisting of 1873 specific proteins of the amphipod crustacean Gammarus fossarum, a species of interest for freshwater biomonitoring. Based on this protein database, the objective of the present study was to develop an approach that allowed a fast, specific and simultaneous quantification of multiple proteins of interest in this species. Following recent methodologies for the development of biomarkers in human disease diagnosis, we implemented a quantitative multiplexed targeted proteomics assay (using Selected Reaction Monitoring mass spectrometry) to study 55 protein biomarker candidates. Identification of specific proteotypic peptides and assessment of their interest as biomarkers in G. fossarum were achieved by following their quantitative changes through male and female reproductive cycles and after exposure to stresses, such as food privation and exposure to contamination through laboratory and in situ (caged organisms) assays. The levels of 21 biomarkers of interest (sex-specific proteins and/or with key physiological functions) were simultaneously monitored in several biological samples during their physiological processes. Their sensitivity to toxic contamination was demonstrated both in laboratory and during field monitoring. For example, the laboratory contamination with environmental concentrations of cadmium modulated the expression levels of some biomarkers annotated as the Na+K+ATPase (cellular pump), catalase (oxidative defense) and GST (detoxification) proteins. This breakthrough methodology in ecotoxicology offers a valid alternative to the currently used protocols, paving the way for future practical applications of protein biomarkers in chemical risk assessment and environmental monitoring.