In this work, analysis of spectra with artificial neural network (ANN) and estimation of glucose concentration from the spectrum is aimed by using near infrared spectroscopy (NIR) method. For this purpose, samples of different concentrations obtained by mixing pure water with glucose monohydrate, which was measured via precision scale, and then NIR spectrum of each sample was extracted. Spectral data and concentration values were transferred to MATLAB platform. ANN analysis was conducted after the required signal processing steps. Experimental results are promising in the determination of glucose concentration of any solution by this method including blood sugar.