In this study, the utility of Raman spectroscopy (RS) with chemometric methods for quantification of multiple components in the fermentation process was investigated. Vinegar, the product of a two stage fermentation, was used as a model and glucose and fructose consumption, ethanol production and consumption and acetic acid production were followed using RS and the partial least squares (PLS) method. Calibration of the PLS method was performed using model solutions. The prediction capability of the method was then investigated with both model and real samples. HPLC was used as a reference method. The results from comparing RS-PLS and HPLC with each other showed good correlations were obtained between predicted and actual sample values for glucose (R-2 = 0.973), fructose (R-2 = 0.988), ethanol (R-2 = 0.996) and acetic acid (R-2 = 0.983). In conclusion, a combination of RS with chemometric methods can be applied to monitor multiple components of the fermentation process from start to finish with a single measurement in a short time. (C) 2013 Elsevier Ltd. All rights reserved.