Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-beta and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay. © 2014 The Author(s). Chemicals/CAS: abiraterone, 154229-19-3; arsenic trioxide, 1303-24-8, 1327-53-3, 13464-58-9, 15502-74-6; chlorpromazine, 50-53-3, 69-09-0; gefitinib, 184475-35-2, 184475-55-6, 184475-56-7; geldanamycin, 30562-34-6; triadimefon, 43121-43-3