An analytical method of post-translational modifications in hemoglobin is disclosed. The analytical method comprises the steps of providing a blood comprising the hemoglobin with post-translational modification of nitration, nitrosylation, or oxidation; performing an extraction process to the blood by an organic solvent; quantifying the hemoglobin by a fluorescent spectrometry; hydrolyzing the hemoglobin into a plurality of peptides by an enzyme; and using a nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry to characterize and quantify the post-translational modifications of the hemoglobin.