The advent of high-resolution mass spectrometers coupled with proteomic techniques has facilitated the discovery and characterization of novel viral proteins and the detection of virus-induced changes in the cellular proteome. These advances have enabled a more comprehensive characterization of viral interactions involved in infection and pathogenesis, and allowed the discovery of viral biomarkers. This article focuses on the role of mass spectrometry proteomic techniques to identify and characterize both prospective and verified viral biomarkers, and their implications on the diagnosis of disease.