The application feasibility of in-situ or in-line monitoring of S. cerevisiae ITV01 alcoholic fermentation process, employing Near-Infrared Spectroscopy (NIRS) and Chemometrics, was investigated. During the process in a bioreactor, in the complex analytical matrix, biomass, glucose, ethanol and glycerol determinations were performed by a transflection fiber optic probe immersed in the culture broth and connected to a Near-Infrared (NIR) process analyzer. The NIR spectra recorded between 800 and 2,200 nm were pretreated using Savitzky-Golay smoothing and second derivative in order to perform a partial least squares regression (PLSR) and generate the calibration models. These calibration models were tested by external validation and then used to predict concentrations in batch alcoholic fermentations. The standard errors of calibration (SEC) for biomass, ethanol, glucose and glycerol were 0.212, 0.287, 0.532, and 0.296 g/L and standard errors of prediction (SEP) were 0.323, 0.369, 0.794, and 0.507 g/L, respectively. Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:510-517, 2016