This research was to develop a method for noninvasive and fast blood glucose assay in vivo. Near-infrared (NIR) spectroscopy, a more promising technique compared to other methods, was investigated in rats with diabetes and normal rats. Calibration models are generated by two different multivariate strategies: partial least squares (PLS) as linear regression method and artificial neural networks (ANN) as non-linear regression method. The PLS model was optimized individually by considering spectral range, spectral pretreatment methods and number of model factors, while the ANN model was studied individually by selecting spectral pretreatment methods, parameters of network topology, number of hidden neurons, and times of epoch. The results of the validation showed the two models were robust, accurate and repeatable. Compared to the ANN model, the performance of the PLS model was much better, with lower root mean square error of validation (RMSEP) of 0.419 and higher correlation coefficients (R) of 96.22%. (C) 2017 Elsevier B.V. All rights reserved.