Updated project metadata. Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here we present a rapid and reproducible data dependent proteomics approach for identifying comprehensive molecular signatures of interference with the thyroid system in zebrafish (Danio rerio) embryos as an alternative to animal testing. For this, we have analysed the thyroid hormone thyronin (T3) as model substances for thyroidal activity in a modified zebrafish embryo toxicity test (zFET). These fingerprints allow for a definition of solid biomarkers as tools in screening approaches and for integration in chronic toxicity studies for suspect substances, such as the fish early life-stage test (OECD TG 210).