Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders affecting both humans and animals. TSEs are caused by the infectious agent 'prion', which is poorly characterized and is believed to be composed of the pathological isoform--TSE-associated prion protein (PrP(TSE))--of a physiological, host-encoded protein called cellular prion protein (PrPC). Whereas it is certain that the process of PrP(TSE) formation has a fundamental role in the development of TSE, other aspects, including the mechanism of this process, the nature and the role of the factors involved (related or unrelated to PrP), and the relationship between PrP(TSE) conformations and disease phenotypes remain poorly defined. Different proteomic strategies have been utilized to address these issues. In this ambit, mass spectrometry (MS) has gained a prominent position, with applications that range from the investigation of the molecular pathogenesis to the development of new diagnostic tools. The aim of this review is to outline these advances and to highlight promising avenues to prion research that have been opened by novel MS applications.