Prion diseases, or transmissible spongiform encephalopathies, are progressive, fatal neurodegenerative diseases. There are both human and animal forms of the disease and all are associated with the conversion of a normal host-coded cellular prion protein (PrPC) into an abnormal protease-resistant isoform (PrPSc). Although methodologies are sensitive and specific for postmortem disease diagnosis, the use of PrPSc as a preclinical or general biomarker for surveillance is difficult, due to the fact that it is present in extremely small amounts in accessible tissues or body fluids such as blood, urine, saliva, and cerebrospinal fluid. Recently, amplification techniques have been developed, which have enabled increased sensitivity for PrPSc detection. However, it has recently been reported that proteinase K sensitive, pathological isoforms of PrP may have a significant role in the pathogenesis of some prion diseases. Accordingly, the development of new diagnostic tests that do not rely on PrPSc and proteinase K digestion is desirable. The search for biomarkers (other than PrPSc) as tools for diagnosis of prion diseases has a long history. Ideally biomarkers able to detect all transmissible spongiform encephalopathies, even at preclinical stages of infection are desirable but not yet possible due to the heterogeneity of the disease and lengthy disease progression. Recent advances in neuroproteomics have led to an overwhelming amount of information, which may offer insight on protein–protein interactions. While the amount of data obtained is impressive, the ability to relate it to the disease and validating its usefulness in diagnostic biomarker development remains a formidable challenge.