Commonly used for research studies in the central nervous system, microdialysis has revealed a link between dysregulation of the excitatory neurotransmitter glutamate and ischemia and seizure, however limitations like slow temporal resolution have stalled the advancement of microdialysis as a diagnostic tool. We have developed and extensively characterized an enzyme-based microelectrode array technology for second-by-second in vivo amperometric measurements of glutamate in the mammalian CNS. The current studies demonstrated the ability of a human microelectrode array prototype (Spencer-Gerhardt-2 (SG-2)) to measure tonic and phasic glutamate neurotransmission in the putamen of unanesthetized non-human primates. We also showed that the SG-2 remains functional following sterilization. Ability to monitor dynamic changes in glutamate in real-time may assist the development of clinical algorithms to potentially alert care-providers prior to onset of overt ischemia or seizure, or provide neurosurgeons with second-by-second measurements of rapid changes in extracellular glutamate which could help guide surgical procedures or aid in interventional strategies.