Background: Methamphetamine (MA) is the most popular recreational drug. According to potential neurotoxicity of this agent, it can cause deleterious effects on neural differentiation of embryo, if MA is used during the child bearing period. In recent decades, undifferentiated pluripotent embryo-derived stem cell lines, resembling early embryonic stages, have been used to analyze the toxic effects of components in vitro. Thus, this study aims at assessing toxic effects of MA on embryonic stem cell (ESC)-derived neuronal cells during differentiation in a pharmacological model. Materials ans Methods : ESC line Royan was used throughout this study. The effect of MA on neural differentiation was assessed during two periods, group 1: MA (10, 100, 200,500, 750, 1000 μM concentrations) was added during EB formation, group 2: MA (10, 50, 70, 100, 200, 500 μM concentrations) was added after the generation of neural precursors. Then cells were evaluated for neuronal markers by immunocytochemistry and RT-PCR. One way ANOVA followed by the post hoc test was used to analyze data. Results: The declining in outgrowth of dendrites was observed in neural morphology in a dose dependent manner. The ID50 (Inhibition of neuronal differentiation) of groups 1 and 2 were 130 and 400 μM, respectively. By using RT-PCR, in comparison with MAP2, no significant change was observed in Nestin expression. Conclusions: Our data on neuronal toxicity were consistent with in vivo and in vitro studies. We concluded that ESCs can be used as an efficient model to assess the toxicity of drugs.