Background & Objectives: The purpose of this study is to investigate the effect of midazolam on neural tube development in the early chicken embryo model. Materials & Methods: One hundred twenty specific pathogen-free chick eggs in four groups were enrolled in the study to investigate the neurulation. All of the groups (A, B, C, D) were incubated for 30 hours. Two eggs from each group were sacrificed at 30th hour. One unfertilized egg in each group was detected. In the end of thirty hours, group A (control group) was administered 0.1 ml of saline (0.9 % NaCl), and the other groups were administered same volume of midazolam solutions of 0.05 (group B), 0.3 (group C), and 0.6 (group D) mg/kg, respectively. At the end of 72 hours, all embryos were excised and they underwent pathological examination. One embryo in group B and another in group D were injured during harvesting and they were excluded from the study. Results: Totally 106 embryos were examined. Distribution of the number of the specimens examined in groups A, B, C, D were 27, 26, 27, 26, respectively. Five-micron thick serial sections were cut from paraffin blocks with rotary microtome and stained with hematoxylin - eosin (HE) for light microscopic examination. No neural tube defects were detected in the study group (0 %), (p = 1) Conclusion: Overall results of our study raises the safety issue of midazolam administration in the early period of embryogenesis for neurulation, however further studies and different embryo models whose drug metabolism resembles human are needed to yield any evidence based data on the issue.