Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o-nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.