Major developmental pathways play critical roles in wide array of human pathologies. Chemical genomic screening allows for the discovery of novel tools not only to target known pathway interactors but also to discover new, chemically tractable targets for known pathways. The zebrafish has emerged as a useful model for developmental biology and has been well characterized. The zebrafish represents a hardy conglomerate of totipotent cells that are massively and simultaneously assessing all significant pathways in parallel in an endogenous context. This represents a gold standard for biological assays, chemically targeting select pathways without causing pleiotropic effects. Here, we describe methods used to develop high content screening assays implementing transgenic zebrafish to assess phenotypic changes that have identified several classes of novel compounds that effect developmental pathways.