Protein tyrosine nitration (PTN) is a posttranslational modification resulting from oxidative/nitrosative stress that has been implicated in a wide variety of disease states. Characterization of PTN is challenging due to several factors including its low abundance in a given proteome, preferential site modification, multiple target site proximity within unique peptide sequences, and analytical method and instrument limitations. Current analytical techniques are insufficiently sensitive to identify endogenous nitration sites without incorporation of either nitrotyrosine or target protein enrichment. However, enrichment proficiency can also be inadequate. Chemical derivatization of the nitro- moiety can be incomplete or result in undesirable byproduct formation, while immunoaffinity proficiency is contingent upon antibody specificity. To overcome analytical method and enrichment deficiencies, we aimed to develop a comprehensive nitroproteome-specific workflow using molecular methods combined with mass spectrometry. Our approach was to systematically address all relevant factors contributing to PTN such as primary sequence, protein conformation, solvent accessibility, and nitrating agent concentration. Our ultimate goal was to increase mass spectrometric sensitivity for PTN identification. All putative nitroprotein/nitropeptide identifications were then subjected to rigorous validation by either manual spectrum analyses or peptide synthesis. We further developed MS methods for quantitation of nitropeptides from complex mixtures with minimal sample processing. Successful application of our nitroproteome-specific mass spectrometric workflow is expected to provide powerful tools for comprehensive PTN investigation that will elucidate its role in the onset and progression of a variety of disease states as well as facilitate discovery of therapeutic targets.