Glycation has been implicated in cataract formation. Our earlier studies showed that crystallin glycation enhances oxidation and aggregation, whereas MIP26 glycation affects the membrane permeability. Scheimpflug densitometric analysis has been used to quantify the lens opacification. In this study, we measured the progressive changes in lens opacification and correlated them with protein glycation in streptozotocin-induced diabetic rats. The lens opacification progressed in a biphasic manner: an initial slow increase between 0 and 60 days, followed by a steep increase between 60 and 90 days of diabetes. There was a strong correlation between lens opacification and lens crystallin and MIP26 glycation. The correlation was relatively weak with plasma glucose. This study suggests that glycation of lens crystallin and MIP26 plays a significant role in the development of lens opacification in diabetic rats.