Teratogens play a crucial role in the development of birth defects, making effective screening vital for prevention and management. This study aimed to develop an optimized zebrafish embryo-based platform for teratogenicity screening and further evaluate its findings with established clinical and animal data. Zebrafish embryos [6-8 h post-fertilization (hpf)] were exposed to 19 different test solutions, including nine known teratogens and ten non-teratogens, in 96-well plates, and mortality and morphological abnormalities were assessed at 48, 72, and 96 hpf. The half-lethal concentration (LC