Induction of vitellogenin (Vg) in males or juveniles is a well known effect of xenoestrogenic contaminants in fish, and has been extensively used as a specific endocrine disruption biomarker. Despite their obvious ecological importance, relatively few works have been carried out on invertebrates and consequently few tools are available to diagnose an endocrine disruptor exposure in these species. At present, only indirect methods, such as the organic alkali-labile phosphate (APL) assay, have been proposed and applied in invertebrates species. From an analytical point of view, efforts should also be directed toward developing more specific methods to measure Vg levels in invertebrates. This study focuses on the development of a new Vg quantitative assay in an ecologically relevant freshwater invertebrate, Gammarus fossarum, using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this end, we followed an approach in three steps, i) we developed a quantitative SRM (Single Reaction Monitoring) assay for the detection of proteotypic Vg peptide, ii) the specificity of this proteotypic peptide as indicator of the functional Vg was validated, by assessing its natural variability during the reproductive cycle of female organisms and iii) the use of this approach as biomarker was evaluated, by studying the modulation of the proteotypic peptide in gammarids exposed to known endocrine disruptors. From this study, an analytical method allowing an absolute quantification of vitellogenin in G. fossarum was developed. The use of this assay as a specific endocrine disruption biomarker was validated by assessing the modulation of vitellogenin levels in gammarids females exposed to the 20-hydroxyecdysone and the methyl-farnesoate.