The number of glycation sites of ovalbumin was monitored by Fourier transform ion cyclotron mass spectrometry (FTICR-MS) before and after reducing the pair of the intrachain disulfide bond. Reducing the disulfide bond of the protein greatly improved the reactivity of glycation both in dry-state and solution. The glycation sites identified by MS/MS showed that the major glycation sites of the ovalbumin were lysines. Our results suggest that glycation is strongly dependent on the protein tertiary structure, with significantly stronger reaction when the protein tertiary structure is disrupted after reducing the disulfide bond. The number of glycated sites of the protein was increased from seven to twelve in dry-state and one to two in aqueous solution. The glycation sites were found to be regulated by protein tertiary structure, hydrogen bonding, and neighboring amino acid compositions.