To detect adverse effects of toxic substances on neurons, we quantitated neuron number, dendrite length and synaptic status of cultured neurons. An actin-binding protein, drebrin accumulated in the postsynaptic sites of glutamatergic synapses and a tubulin-binding protein, MAP2 were used as markers to detect synaptic changes and to visualize neuronal cell body and dendrites, respectively. We have applied this method for high-throughput analysis and showed that glutamate treatment for 10 min significantly reduced drebrin cluster density of 21-days-in-vitro (DIV) neurons in a dose-dependent manner. In this study, we examined the effects of other toxic substances. Treatment of 0.5-50 µM latrunculin A, which sequesters monomeric actin, for 5 min significantly reduced drebrin cluster density of 21-DIV neurons in a dose-dependent manner. We also confirmed that exposure of 1 Gy X-irradiation to 1-DIV neurons reduces neuron number, dendrite length and drebrin cluster density in the neurons at 21-DIV. In addition, our analysis could efficiently detect staurosporine-induced neuronal cell death in mature neurons. 24 hours exposure of 0.3 and 1.0 µM staurosporine to 21-DIV neurons significantly reduced neuron number. These results suggest that our high-content imaging analysis is useful for analyzing the effects of various toxic substances.