High-dose methotrexate (MTX) application is widely used for the treatment of acute lymphoblastic leukemia in children. MTX can cross blood-brain barrier to reach neurons when applied with a high dose. Indeed, high-dose MTX application could cause neurological side effects including cognitive impairment. In our previous reports, we established a high-throughput imaging system for the detection of the effects of various neurotoxic compounds using primary cultured neurons. Using this system, we studied the neurotoxic effects of various concentrations of MTX in the present study. For this purpose, primary cultured cerebral cortical cells were prepared from embryonic days 17 Wister rat. Cerebral cortical cells were cultured for 21 days on 96-well microplates. At 4 days in vitro (4 DIV), MTX was applied to the cultured cells in each well at the concentration of 0.01, 0.03, 0.1, 0.3, 1, 10, or 100 µM, respectively. At 21 DIV, cerebral cortical cells were fixed with paraformaldehyde and stained with DAPI and with neuronal and synaptic markers. To our surprise, low concentrations of MTX reduce the number of cultured cerebral cortical neurons in the well. Our finding indicates that MTX could be toxic at a concentration lower than what we expected.