AbstractOxidative stress plays important roles in a wide range of diseases such as cancer, inflammatory disease, neurodegenerative disorders, etc. Tyrosine nitration in a protein is a chemically stable oxidative modification, and a marker of oxidative injuries. Mass spectrometry (MS) is a key technique to identify nitrotyrosine‐containing proteins and nitrotyrosine sites in endogenous and synthetic nitroproteins and nitropeptides. However, in vivo nitrotyrosine‐containing proteins occur with extreme low‐abundance to severely challenge the use of MS to identify in vivo nitroproteins and nitrotyrosine sites. A preferential enrichment of nitroproteins and/or nitropeptides is necessary before MS analysis. Current enrichment methods include immuno‐affinity techniques, chemical derivation of the nitro group plus target isolations, followed with tandem mass spectrometry analysis. This article reviews the MS techniques and pertinent before‐MS enrichment techniques for the identification of nitrotyrosine‐containing proteins. This article reviews future trends in the field of nitroproteomics, including quantitative nitroproteomics, systems biological networks of nitroproteins, and structural biology study of tyrosine nitration to completely clarify the biological functions of tyrosine nitration. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 34: 423–448, 2015.