Drebrin is a major F-actin binding protein in dendritic spines that is critically involved in their morphological plasticity. Subcellular localization of drebrin is dependent on NMDAR activity. Drebrin's change in dendritic spines is an indicator of toxic effect of compounds to the brain and can be used for prediction of brain dysfunction prior to the neuronal cell death. In the present study, we have developed new drebrin clusters analysis method applied the confocal high-content screening method. Hippocampal neurons prepared from embryonic rats (SKY neuron, AlzMed, Inc., Tokyo) were incubated in 96-well microplates. After 21 days, the cultured neurons were treated with 10, 100 µM glutamate. After the treatments, they were fixed and processed for immunocytochemistry to visualize drebrin, MAP2 and cell nucleus. After automated image acquisitions, neuron number, dendrite length, drebrin clusters were quantified with original algorithm. The high-throughput immunocytochemical assay demonstrated that glutamate treatment decreased drebrin cluster density. The ratio of decrease in the density was concentration dependent and found to be dependent on the number of surrounding neurons. Our method is sensitive enough to detect the interaction with NMDAR and is useful for drug screening studies for synaptic dysfunction.