Core endpoints in zebrafish embryos are crucial indicators in screening harmful effects of chemicals. In this study, we established a three-step process to more quantitatively and less-subjective determine effects of chemicals on phenotypes of developing zebrafish embryos. Embryos were exposed to each of two concentrations of the representative chemicals cadmium chloride (CdCl2), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) or 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH) from 0.75 h post-fertilization (hpf) to 96 hpf. After exposure, larvae were imaged by use of a three-step method to describe morphology. Seven points were selected, which resulted in acquisition of 21 lines and 105 angles from images of larvae. Exposure to TDCIPP (0.1 or 0.2 mg/L), CdCl2 (1 or 4 mg/L) or 4:2 FTOH (0.3 or 1 mg/L) significantly changed lengths of some lines and magnitudes of some angles, that resulted in differential scoring of points. Points were then prioritized and directions, distances and trajectories of movement were further described and standard reference values were developed. Movement of the point describing the mouth during embryonic development was found to be a sensitive parameter for assessment of adverse effects of chemicals. The present study provides a new strategy to characterize phenotypes of development of zebrafish embryo/larva following exposure to environmental toxins. (C) 2017 Elsevier Ltd. All rights reserved.