Glutamate is involved in excitatory neurotransmission and oxidative metabolism. In vivo glutamate measurements at task-relevant temporal resolution are possible using functional proton magnetic resonance spectroscopy. The present study quantified glutamate levels in the dorsolateral prefrontal cortex during working memory task performance and interspersed periods of rest. Results demonstrated that our approach was feasible and quantification of glutamate levels was reliable at 32s temporal resolution. Statistical analyses demonstrated that glutamate levels were elevated during working memory task performance (relative to rest), consistent with our hypotheses. Elevated glutamate levels during task performance likely reflect increased oxidative metabolism due to excitatory neurotransmission.