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In vitro neurotoxicity testing has been hampered by the fact that the brain architecture is complex. However, a series of innovation of neuroculturing broke through the barrier. The establishment of culturing for the primary neuron and the immobilised neuroblastoma cells enables neurotoxicity testing in vitro. Following to necrotic cell death, extensive morphological changes as seen during neuronal differentiation was used for the endpoints of neurotoxicity. Two-dimensional imaging techniques facilitated quantitative analyses of toxicity of many neutoxicants. Three-dimensional culturing of neurospheres in vitro has been expected to investigate the neurodevelopmental toxicity. The neurosphere assay in vitro also improved the sensitivity to estimate the neurotoxicity. The present study highlights an overview about the in vitro neurotoxicity testing for rotenone, a dopaminergic pesticide as an environmental toxicant.