In the human brain, glial cells such as astrocytes, microglia, and oligodendrocytes interact with neurons to form a microenvironment. Astrocytes, a type of glial cells, promote the formation of synapses through which nerve cells extend axons and transmit electrical signals, and stabilize structures. We tried to develop a neurotoxicity evaluation model that can consider cell-cell interactions in a neuron co-culture system that mimics the human brain environment. Based on the cell morphology and immunostaining characteristics confirmed in the co-culture conditions of human neuroblastoma SH-SY5Y cells and human iPS cell-derived astrocytes, co-culture conditions suitable for observing neurites were determined. In order to verify the established co-culture model as a neurotoxicity evaluation model, the effect on neurodevelopment was evaluated using well-known neurotoxic substances, acrylamide and hydrogen peroxide. Quantitative analysis of neurite outgrowth and cell nuclei using high-content screening showed that the neurotoxin significantly inhibited neurite outgrowth, a hallmark of neurodevelopment. Further analysis confirmed that the neurotoxins modulate genes involved in neurodevelopment. Comparative analysis between SH-SY5Y and co-culture showed that astrocytes have neuroprotective effects on neurons. Our neurotoxicity assessment model can propose an efficient neural-based in vitro neurotoxicity assessment method and illustrate the advantages of considering the cell-cell interactions of the human brain microenvironment.