© 2018 Elsevier Inc. The aim of this study was to evaluate if glucose, fructosamine, and insulin levels can be measured in saliva of dogs and assess the changes in these compounds after an experimental glucose administration. Automated spectrophotometric assays for glucose and fructosamine and an ELISA assay for insulin measurements were validated in saliva of dogs, by evaluating precision, accuracy, and limits of detection. In addition, an intravenous glucose bolus was administrated to 10 beagles and fasting serum and saliva samples were obtained immediately before and 5, 10, 20, 30, and 45 min after glucose infusion. The results of the between-run imprecision gave mean CVs of 6.16, 9.40, and 3.10% for glucose, fructosamine, and insulin, respectively. Linearity under dilution showed coefficient of correlation of 0.999, 0.994, and 0.990 for glucose, fructosamine, and insulin, respectively. The LDs were 0.04 mg/dL, 4.08 μmol/L, and 0.02 μg/mL for glucose, fructosamine, and insulin, respectively. The glucose administration caused an increase in serum and salivary levels of glucose with a peak in salivary levels at 30 min and of insulin with a peak in salivary levels at 45 min, while fructosamine did not change. No correlations between serum and salivary concentrations were found for any compound. It is concluded that glucose, fructosamine, and insulin can be measured in saliva of dogs, and an experimental administration of glucose in this species can lead to increases in glucose and insulin in saliva.