Direct electrochemical measurements of glutamate release in vivo were combined with optogenetics in order to examine light-induced control of glutamate neurotransmission in the rodent brain. Self-referenced recordings of glutamate using ceramic-based microelectrode arrays (MEAs) in hippocampus and frontal cortex demonstrated precise optical control of light-induced glutamate release through channelrhodopsin (ChR2) expression in both rat hippocampus and frontal cortex. Although the virus was only injected unilaterally, bilateral and rostro-caudal expression was observed in slice imaging, indicating diffusion and active transport of the viral particles. Methodology for the optogenetic control of glutamate signaling in the rat brain is thoroughly explained with special attention paid to MEA enzyme coating and cleaning for the benefit of other investigators. These data support that optogenetic control of glutamate signaling is robust with certain advantages as compared to other methods to modulate the in vivo control of glutamate signaling.