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Monitoring of blood glucose is an invasive, painful and costly practice in diabetes. Consequently, the search for a more cost-effective (reagent-free), non-invasive and specific diabetes monitoring method is of great interest. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used in diagnosis of several diseases, however, applications in the monitoring of diabetic treatment are just beginning to emerge. Here, we used ATR-FTIR spectroscopy to evaluate saliva of non-diabetic (ND), diabetic (D) and diabetic 6U-treated of insulin (D6U) rats to identify potential salivary biomarkers related to glucose monitoring. The spectrum of saliva of ND, D and D6U rats displayed several unique vibrational modes and from these, two vibrational modes were pre-validated as potential diagnostic biomarkers by ROC curve analysis with significant correlation with glycemia. Compared to the ND and D6U rats, classification of D rats was achieved with a sensitivity of 100%, and an average specificity of 93.33% and 100% using bands 1452 cm−1 and 836 cm−1, respectively. Moreover, 1452 cm−1 and 836 cm−1 spectral bands proved to be robust spectral biomarkers and highly correlated with glycemia (R2 of 0.801 and 0.788, P < 0.01, respectively). Both PCA-LDA and HCA classifications achieved an accuracy of 95.2%. Spectral salivary biomarkers discovered using univariate and multivariate analysis may provide a novel robust alternative for diabetes monitoring using a non-invasive and green technology.