The function and activity of almost every circuit in the human brain are modified by the signaling peptides (SPs) surrounding the neurons. As the complement of peptides can vary even in adjacent neurons and their physiological actions can occur over a broad range of concentrations, the required figures of merit for techniques to characterize SPs are surprisingly stringent. In this review, we describe the formation and catabolism of SPs and highlight a range of mass spectrometric techniques used to characterize SPs. Approaches that supply high chemical information content, direct tissue profiling, spatially resolved data, and temporal information on peptide release are also described. Because of advances in measurement technologies, our knowledge of SPs has greatly increased over the last decade, and SP discoveries will continue as the capabilities of modern measurement approaches improve.