Neuropeptides are intercellular signal transmitters that play key roles in modulation of many behavioral and physiological processes. Neuropeptide signaling in several nuclei in the hypothalamus contributes to the control of food intake. Additionally, food intake regulation involves neuropeptide signaling in the reward circuitry in the striatum. Here, we analyze neuropeptides extracted from hypothalamus and striatum from rats in four differentially treated dietary groups including a high-fat/high-sucrose diet, mimicking diet-induced obesity. We employ high-resolution tandem mass spectrometry using higher-energy collision dissociation and electron transfer dissociation fragmentation for sensitive identification of more than 1700 unique endogenous peptides, including virtually all key neuropeptides known to be involved in food intake regulation. Label-free quantification of differential neuropeptide expression revealed comparable upregulation of orexigenic and anorexigenic neuropeptides in rats that were fed on a high-fat/high-sucrose diet.