The kinetic procedure of the unfolding of lysozyme induced by the reduction of disulfide was monitored by the time-resolved ESI-MS with a sheath liquid assistant electrospray interface. It was found that the reduction process for the eight disulfides had a less difference in the reaction time after denatured treatment. In addition, the alkylation of the reduced free thiols was much slower than the reduction procedure. An artifact peak produced by the CID fragmentation in the mass spectra was identified and the possible mechanism of the Hofmann elimination reaction was proposed.