Neuropeptides are critical signaling molecules, involved in the regulation of diverse physiological processes including energy metabolism, pain perception and brain cognitive state. Prolonged general anesthesia has an impact on many of these processes, but the regulation of peptides by general anesthetics is poorly understood. In this study, we present an in-depth characterization of the hypothalamic neuropeptides of the tree shrew during volatile isoflurane/nitrous oxide anesthesia administered accompanying a neurosurgical procedure. Using a predicted-peptide database and hybrid spectral analysis, we first identified 85 peptides from the tree shrew hypothalamus. Differential analysis was then performed between control and experimental group animals. The levels of 12 hypothalamic peptides were up-regulated following prolonged general anesthesia. Our study revealed for the first time that several neuropeptides, including alpha-neoendorphin and somatostatin-14, were altered during general anesthesia. Our study broadens the scope for the involvement of neuropeptides in volatile anesthesia regulation, opening the possibility for investigating the associated regulatory mechanisms.