Neuroimaging and electrophysiological studies have consistently provided evidence on the role of dorsolateral prefrontal cortex in working memory. Recent studies have shown that the changes in metabolites in the brain could be detected upon functional activation using proton magnetic resonance spectroscopy. Therefore, we carried out a study in healthy control participants to understand the changes in metabolites during a letter N-back working memory task in the left dorsolateral prefrontal region. Single-voxel spectra were acquired from the left dorsolateral prefrontal region in 10 healthy control participants. The concentrations of metabolites were measured using a point-resolved spectroscopy sequence at three different time points, namely, before, during, and after the performance of the task. Repeated-measures analysis of variance indicated a main effect of time for glutamate concentrations and not for other metabolites. The healthy control participants showed a significant increase in glutamate concentrations during the performance of the letter N-back task. It was then followed by a significant decrease in the glutamate concentrations returning to the baseline after the task. The proposed method would be useful in understanding the dynamic changes in glutamate during the letter N-back task to examine the cognitive impairment in various neurodegenerative and psychiatric disorders.