The application described in this paper addresses problems encountered during the large scale production of ethanol from renewable sources. Bioethanol can be produced from several starchy raw materials (i.e. wheat, corn, barley, rye). After hydrolysis to glucose, starch can be converted in ethanol by fermentation. The hydrolysis of starch requires a multi-phase process using different enzymes. The degree of hydrolysis is a critical parameter because it determines the physical and chemical properties of the final products. The aims of this work were to monitor the enzymatic hydrolysis of starch from rye using Fourier-transformed near infrared (FT-NIR) spectroscopy, and to identify barriers for an industrial scale application of this technology. The enzymatic hydrolysis of starch from rye was carried out in three steps, using commercially-available enzymes. A FT-NIR reflectance probe was immersed in the reactor to monitor the different phases of the hydrolysis process. The most significant parameters submitted to the NIR calibration were the percentage of dry matter (%w/v) (degreesBrix) and the concentrations of maltotriose, maltose and glucose (% w/v). PLS calibration models were built using 24 samples and spectra fordegreesBrix, 32 samples for maltotriose, and 48 samples for both maltose and glucose, respectively. The models were considered as promising in terms of the squared Pearson correlation coefficient (0.89
