This study aimed to investigate the effects of valproate on the levels of cerebral amino acid neurotransmitters during K+-evoked cortical spreading depression (CSD) in male Wistar rats (200-300 g) using microdialysis technique. The rats were anesthetized by urethane (1.5 g/kg, i.p.) and then placed in stereotaxic frame. The scalps were cut and opened to expose the skulls and two burr holes were drilled on the right hemisphere. The anterior hole at the frontal bone was to place microdialysis probe (aCSF, 2 µl/min flow rate), whereas the posterior hole at the parietal bone was used for an application of solid KCl (3 mg). Microdialysis probe was inserted into frontoparietal cortex and intraperitoneal injection of either 0.5% Carboxymethyl methyl cellulose sodium (1 ml/kg) or valproate (200 mg/kg), was made following a stabilization period of 60 minutes. Thirty minutes after administration of the test compounds, CSD was induced by placing solid KCl onto the brain and observation was made for another 90 minutes. Dialysates were collected every 30 minutes throughout the experimental period and being analyzed for amino acid neurotransmitters by High Performance Liquid Chromatography with Fluorescent Detection. It was found that levels of both cortical excitatory (glutamate and aspartate) and inhibitory (GABA and glycine) amino acid neurotransmitters in VPA-treated group were significantly decreased in comparison to those of CMC-treated group at any observed times (p = 0.05). However, the depression was greatest on glutamate which is a major excitatory amino acid neurotransmitter whereas smaller effect was noted on the others. The results obtained from this study were consistent with electrophysiological depression of CSD by VPA previously reported by other investigators and may explain the effect of VPA in migraine prophylaxis.