Traumatic stress triggers or exacerbates multiple psychiatric illnesses, including post-traumatic stress disorder (PTSD). Nevertheless, the neurophysiological mechanisms underlying stress-induced pathology remain unclear, in part due to the limited understanding of neuronal signaling molecules, such as neuropeptides, in this process. Here, we developed mass spectrometry (MS)-based qualitative and quantitative analytical strategies to profile neuropeptides in rats exposed to predator odor (an ethologically relevant analogue of trauma-like stress) versus control subjects (no odor) to determine peptidomic alterations induced by trauma. In total, 628 unique neuropeptides were identified across 5 fear-circuitry-related brain regions. Brain-region-specific changes of several neuropeptide families, including granin, ProSAAS, opioids, cholecystokinin, and tachykinin, were also observed in the stressed group. Neuropeptides from the same protein precursor were found to vary across different brain regions, indicating the site-specific effects of predator stress. This study reveals for the first time the interaction between neuropeptides and traumatic stress, providing insights into the molecular mechanisms of stress-induced psychopathology and suggesting putative novel therapeutic strategies for disorders such as PTSD.