Glycation is a non-enzymatic process between the reactive carbonyl group of sugars and free amino groups of proteins especially arginine and lysine residues. This process leads to the formation of a group of compounds called as Amadori products and advanced glycation products. These products have been implicated in many secondary complications of diabetes. In the last few years, the intake of sweetener has increased for various health reasons like control of hyperglycaemia and obesity. The present study was designed to evaluate the effect of Acesulfame-K, a well-known and widely used sweetener, on glycation system of arginine-glucose and lysine-glucose. The number of glycation products generated in the presence and absence of acesulfame potassium was measured by established methods such as browning, Fructosamine assay, and determination of carbonyl content. The effect of acesulfame potassium was also checked on the glycation of DNA by agarose gel electrophoresis method. The results indicate that lysine is more potent in causing glycation as compared to arginine. Acesulfame potassium could significantly decrease the number of glycation products in the glycation systems, arginine-glucose and lysine-glucose. It can be concluded that Acesulfame-K has anti-glycation potential as it decreased the formation of Amadori products and AGEs. This study is significant in understanding the role of artificial sweetener in the process of glycation.