West Nile Virus (WNV) was introduced into North America in 1999 where it has emerged as the most common cause of arboviral neuroinvasive disease. An estimated 2 million infections occurred between 1999 and 2013 in North America and there is no specific treatment or vaccine available. WNV-infected cells can be detected and destroyed by CD8+ cytotoxic T lymphocytes via the presentation of viral peptides by major histocompatability complex class I molecules. Briefly, class I MHC molecules provide CD8+ cytotoxic T lymphocytes of the immune system with a “window” into the cell by sampling peptides from all protein constituents within a cell and then displaying these peptides at the plasmalemma. Currently, no one knows how many WNV derived peptides decorate the MHC of infected cells, nor do we know which viral proteins MHC molecules sample. Understanding the number and origin of viral peptides available for immune recognition is a key prerequisite in the development of immunotherapeutics that target intracellular disorders. Using secreted HLA class I molecules in a comparative proteomics system, we eluted peptide ligands from infected and uninfected class I molecules and compared these ligands by mass spectrometry. The resulting data demonstrate that different class I MHC molecules consistently prune a Flavivirus of more than 3,000 amino acids down to a handful of peptides for immune recognition. We then extended this experimental protocol to HIV-1 infected cells, influenza infected cells, breast cancer cells, and ovarian cancer cells. In all instances we find a dozen or fewer MHC class I presented peptide ligands distinguish the diseased from the healthy cell. Finally, peptide based vaccines as well as antibodies specific for peptide/MHC complexes demonstrate the therapeutic potential of these peptide/MHC complexes during infection and cancerous transformation.