Advanced glycation end products (AGEs) can be caused during a glycoxidation reaction. This reaction is asso-
ciated with complications of diabetes and the consequences of health problems. Therefore, we are exploring the
prohibitory effect of highland barley protein hydrolysates (HBPHs) on AGE formation. Herein, first extracted the
protein from highland barley with various pH conditions and then hydrolyzed using four different proteolytic
enzymes (flavourzyme, trypsin, papain, pepsin) under different degrees of hydrolysis. We assessed three degrees
of hydrolysates (lowest, middle, highest) of enzymes used to characterize the antioxidant activity and physi-
cochemical properties. Among all the hydrolysates, flavourzyme-treated hydrolysates F-1, F-2, and F-3 indicated
the high ability to scavenge DPPH (IC50 values of 0.97 %, 0.63 %, and 0.90 %), structural and functional
properties. Finally, the inhibitory effect of the most active hydrolysates F-1, F-2, and F-3 against the AGEs for-
mation was evaluated in multiple glucose-glycated bovine serum albumin (BSA) systems. Additionally, in a BSA
system, F-3 exhibited the strong antiglycation activity, effectively suppressed the non-fluorescent AGE (CML),
and the fructosamine level. Moreover, it decreased carbonyl compounds while also preventing the loss of thiol
groups. Our results would be beneficial in the application of the food industry as a potential antiglycation agent
for several chronic diseases.