We have previously found that glyceraldehyde-derived advanced glycation end products (glycer-AGEs) elicit oxidative stress generation and evoke inflammatory and thrombotic reactions through their higher binding affinity to RAGE (receptor for AGEs), thereby playing a role in vascular complications in diabetes. Furthermore, circulating levels of glycer-AGEs are elevated in diabetes. We characterized a monoclonal antibody (mAb) raised against glycer-AGEs and prepared its specific ELISA system in human serum. We developed here mAb reacted specifically with glycer-AGEs or glyceraldehyde-derived pyridinium, but not other structurally identified AGEs or AGE precursors. The mAb not only completely neutralized the deleterious effects of glycer-AGEs on endothelial cells, but also detected glycer-AGEs in the aorta of type 2 diabetic rats. Intra and inter-assay coefficient variations of the ELISA were 6 and 2.6%, respectively. ELISA linearity was shown intact within 5-fold dilution, and recovery ratio of added glycer-AGEs was 88-117%. Results of serum and plasma were comparable, and repeated freeze-thawing of samples did not affect the results (90.1-112.4%). Serum glycer-AGEs levels in 30 healthy subjects evaluated by the ELISA were strongly correlated with those by polyclonal Ab-based one (r=0.82). Our present study suggests the clinical utility of mAb for evaluating glycer-AGE levels in both tissue and serum.