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Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Arantza Muriana reports equipment, drugs, or supplies and writing assistance were provided by GAIKER. Arantza Muriana reports a relationship with GAIKER that includes: consulting or advisory. The author is co-founder and R&D Director of BBD BioPhenix SLU, and all the coauthors except Sainz L and De Goñi F are nowadays employed or have been employed previously by BBD BioPhenix SLU, a CRO specialized in the use of zebrafish for efficacy, toxicity and ecotoxicity assays, including offering teratogenicity, reprotoxicity, neurotoxicity, or endocrine disruption assays for Pharma Industry, Cosmetic Industry, or Agrochemical Industry, including the assays described in the paper. Sainz L and De Goñi F work in GAIKER, a company that BBD BioPhenix SLU outsources to develop bioavailability assays for their clients, in this case, they offered their services to analyze the compounds inside the zebrafish for this paper., (Copyright © 2023. Published by Elsevier Inc.)