Neuropeptides play a crucial role in cell communication as neurotransmitters, neuromodulators or neurohormones, and are involved in a number of biological activities including neuroendocrine regulations, control of neurovegetative functions, trophic effects and modulation of the immune response. The number of neuropeptides that have been fully characterized so far is rather limited, as compared to the number of precursor proteins that are actually expressed in nerve cells. Owing to the development of powerful analytical and structural identification methods, and the rapid advance in molecular biology techniques, a number of novel neuropeptides have been characterized during the last decade, in both vertebrates and invertebrates. The aim of the present review is to provide a comprehensive coverage of the different approaches which are currently used to identify novel neuropeptides.