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Cost-effective methodologies along with cross-species applicability constitute key points for biomarker development in ecotoxicology. With the advent of cheaper affordable genomic techniques and high throughput sequencing, omics tools could facilitate the assessment of effects of environmental contaminants for all taxa biodiversity. We assessed the potential of absolute quantification of proteins using mass spectrometry to develop vitellogenin (Vg)-like protein assays for invertebrates. We used available sequences in public databases to rapidly identify Vg-proteotypic peptides in seven species from different main taxa of protostome invertebrates (mollusk bivalves, crustacean amphipods, branchiopods, copepods and isopods, and insect diptera). Functional validation was performed by comparing proteomic signals from reproductive female tissue samples and negative controls (male or juvenile tissues). In a second part, we demonstrate in gammarids, daphnids, drosophilids, and gastropods that the assay validated in Vg-sequenced species can be applied to Vg-unsequenced species thanks to the evolutionary conservation of Vg-proteotypic peptide motifs. Finally, we discuss the relevance of mass spectrometry for biomarker development (specific measurement, rapid development, transferability across species). Our study supplies an illustration of the promising strategy to address the challenge of biodiversity in ecotoxicology, which consists in employing omics tools from comparative and evolutionary perspectives.