Background: Although isotretinoin has been confirmed by clinical and experimental studies to be a teratogenic agent that causes fetal developmental abnormalities, its effects on placental development and placental-like extra-embryonic vascularization have not been sufficiently studied. The vascularization of the chorioallantoic membrane is structurally and functionally similar to mammalian placental vascularization, which provided the rationale for conducting this study. Aim: To study the effect of isotretinoin on chorioallantoic membrane vascularization using an in ovo chick embryo model. Materials and Methods: A comparative experimental control study was conducted using fertilized eggs of the same developmental age from Gallus gallus domesticus. At the HH10 embryonic stage, the control group received avian saline, while the experimental group was administered isotretinoin (5 µg/100 µL). Vascularization of the yolk sac and chorioallantoic membrane was evaluated at the macro- and tissue levels at 72 and 120 hours of embryonic development. Vessel branching order, vascular density, and capillary number were compared, and statistical analysis was performed using the Mann-Whitney U test. Result: In the control group, vascularization progressed actively, with abundant formation of third and fourth order vessels, resulting in a dense reticular network. In contrast, the isotretinoin treated group showed delayed vessel branching, a marked reduction in the number of third- and fourth-order vessels, and an overall decrease in vascular density of 40–60%. Histological analysis revealed thinning of the chorioallantoic membrane, loss of tissue layers, and thinning of vascular walls, and the number of capillaries was significantly lower than in the control group (p<0.01). In addition, the development of chorioallantoic membrane vascularization in chick embryos was found to be structurally and developmentally similar to mammalian placental vascularization. Conclusion Isotretinoin was shown to inhibit the development of the chorioallantoic membrane and markedly reduce vascularization and differentiation in an in ovo chick embryo model.