Two-dimensional (2D) gel electrophoresis allows separation of complex mixtures of proteins based on isoelectric points and relative mobility. This method has not changed much fundamentally since their original description in the late 1970s. Despite several limitations, such as solubilization of membrane proteins and separation of highly basic proteins, this method has been used successfully in many laboratories as part of proteomics protocols. Our laboratory coupled 2D-PAGE with 2D Western blot analysis to identify brain proteins modified oxidatively with excess carbonylation, bound 4-hydroxy-2-nonenal, or 3-nitrotyrosine (3-NT) in various diseases and animal models of these disorders. This chapter describes in detail the protocol used for the identification of 3-NT-modified proteins in biological samples that may help in delineating the role of protein nitration in the progression or pathogenesis of various diseases.