The effects of glycation by glyoxal (Gly) and methylglyoxal (MGly) on the early and late conformational alterations in Cytochrome c (Cyt c) were studied. Spectroscopic measurements revealed that Cyt c undergo certain conformational alterations and exposure of heme upon overnight incubation with Gly and MGly. These were followed by the reduction of heme centre and activation of its peroxidase-like, which is crucial for initiation of the intrinsic apoptotic pathway. An extended incubation resulted in appearance of AGE-like fluorescence, with significant alterations in secondary structural compositions. However, no amyloidogenic conversions were observed as suggested by TEM analyses. The study provides an insight to the role of glycating agents, elevated under oxidative stress in inducing Cyt c release and apoptosis.