More precise identification and treatment monitoring of prediabetic/diabetic individuals will require additional biomarkers to complement existing diagnostic tests. Candidates include hyperglycemia-induced adducts such as advanced glycation end products (AGEs) of proteins, lipids, and DNA. The potential for DNA-AGEs as diabetic biomarkers was examined in a longitudinal study using the Lepr^db/db animal model of metabolic syndrome. The DNA-AGE, N²-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) was quantified by mass spectrometry using isotope dilution from the urine and tissue of hyperglycemic and normoglycemic mice. Hyperglycemic mice (fasting plasma glucose, FPG, ≥ 200 mg/dL) displayed a higher median urinary CEdG value (238.4 ± 112.8 pmol/24 h) than normoglycemic mice (16.1 ± 11.8 pmol/24 h). Logistic regression analysis revealed urinary CEdG to be an independent predictor of hyperglycemia. Urinary CEdG was positively correlated with FPG in hyperglycemic animals and with HbA1c for all mice. Average tissue-derived CEdG was also higher in hyperglycemic mice (18.4 CEdG/10⁶ dG) than normoglycemic mice (4.4 CEdG/10⁶ dG). Urinary CEdG was significantly elevated in Lepr^db/db mice relative to Lepr^wt/wt, and tissue CEdG values increased in the order Lepr^wt/wt < Lepr^wt/db < Lepr^db/db. These data suggest that urinary CEdG measurement may provide a noninvasive quantitative index of glycemic status and augment existing biomarkers for the diagnosis and monitoring of diabetes.