Enkephalins are opioid peptides that modulate analgesia, reward, and stress.In vivodetection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. We present an analytical method for Met- and Leu-Enkephalin detection in the mouse Nucleus Accumbens shell after acute stress. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enkephalin through methionine oxidation, and provides insight into the relationship between Met- and Leu-Enkephalin following stress.