A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the COFRADIC (combined fractional diagonal chromatography) peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Dithionite is here used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides which thereby become more hydrophilic. Our COFRADIC technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified 6 in vivo nitration events in 4 serum proteins thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.