Glutathionylation is an example of reversible post-translation modification of proteins where free and accessible cysteine residues of proteins undergo thiol-disulfide exchange with oxidized glutathione (GSSG). In general, glutathionylation occurs under the condition of elevated oxidative stress in vivo. In human hemoglobin, Cys93 residue of beta globin chain was found to undergo this oxidative modification. Glutathionyl hemoglobin (GSHb) was reported to act as a biomarker of oxidative stress under several clinical conditions such as chronic renal failure, iron deficiency anemia, hyperlipidemia, diabetes mellitus, Friedreich's ataxia, atherosclerosis. Previously we showed that the functional abnormality associated with six-fold tighter oxygen binding of GSHb supposedly attributed to the conformational transition of the deoxy state of GSHb towards oxy hemoglobin like conformation. In the present study, we investigated the structural integrity and overall architecture of the quaternary structure of GSHb using native mass spectrometry and ion mobility mass spectrometry platforms. The dissociation equilibrium constants of both tetramer/dimer (K-d1) and dimer/monomer equilibrium (K-d2) was observed to increase by 1.91 folds and 3.64 folds respectively. However, the collision cross-section area of the tetrameric hemoglobin molecule remained unchanged upon glutathionylation. The molecular dynamics simulation data of normal human hemoglobin and GSHb was employed to support our experimental findings.