Neuropeptidomics, a mass spectrometry-based technique which aims to uncover the complete suite of neuropeptides present in a tissue, organ or cell from the brain or nervous system, has found application in studies examining physiological responses (e.g. food intake, appetite and reproduction). Neuropeptides (and peptide hormones) have long been known as regulators of mammalian physiological processes, particularly reproduction. These peptides are derived from precursor proteins and become active via proteolytic processes and post-translational modifications. A relatively large number of neuropeptides, mainly formed in the hypothalamus or the anterior pituitary of mammals, have been specifically associated with reproduction, including GnRH, NPY, PYY and kisspeptin. Here, we will present an overview of neuropeptides, their roles in reproduction and the application of neuropeptidomics in this field. We address the advantages of neuropeptidomics in reproductive studies including the high throughput identification, profiling and quantification of neuropeptides present in reproductive tissues and also discuss some of the challenges. The application of neuropeptidomics to the field of reproduction will provide the foundation for a greater understanding of how neuropeptides act to regulate reproductive function.