The immunohistochemical detection of nitrotyrosine is a robust method for detecting peroxynitrite and other reactive nitrogen species. Success depends on optimizing conditions for the particular tissue and experimental design under investigation and the use of positive and negative controls to verify specificity. The two controls of dithionite reduction and blocking with nitrotyrosine are a powerful combination to demonstrate specificity. The pathological significance of tyrosine nitration in proteins can also be approached. Generally, nitrated proteins can be isolated from diseased tissues by immunoprecipitation and Western blotting. The sites of nitration on specific proteins can be determined by mass spectrometry, which has revealed surprising specificity in which tyrosines and/or proteins are nitrated in vivo. This provides important evidence concerning the functional consequences of peroxynitrite formation in vivo.