3-Nitrotyrosine (3-NT) is used as a biomarker of nitrosative stress. The formation of 3-NT has been reported in a number of diseases, including Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and cancer. The nitration of proteins is a reversible process, but it can induce a conformational change and thereby functional alterations of the affected protein. 3-NT measurements in biological samples are usually carried out by methodologies such as immunohistochemistry, high-performance liquid chromatography, gas chromatography, and immunochemical detection. This chapter describes the immunochemical method for the determination of protein-bound 3-NT using slot-blot analysis.