Within Europe, alternative methods for toxicity assessment of xenobiotics become very important. Several pharmaceutical, agrochemical and cosmetic companies are currently using the zebrafish embryo as an alternative for animal testing to screen new compounds for developmental toxicity. However, false negative and false positive results are reported in the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) for known mammalian teratogens and non-teratogens, respectively. From a safety point of view, false negative results are more critical because teratogens may be missed by the assay. This project aims to further refine the ZEDTA protocol in order to increase the sensitivity of the assay and to advance the ZEDTA from screening towards potential regulatory acceptance. In order to achieve this goal, first the number of evaluated morphological endpoints will be extended by including skeletal staining in the ZEDTA. Second, due to the intrinsically low biotransformation capacity of zebrafish embryos, a metabolic activation system will be developed in order to expose the zebrafish embryos not only to the parent compound but also to its potential teratogenic metabolite(s). Finally, additional morphological endpoints will be programmed in image analysis software that will be combined with an automated handling and imaging system. As such, this project will increase the sensitivity and throughput of the ZEDTA for potential regulatory use.