Soft sensors are powerful tools for bioprocess monitoring due to their ability to perform online, noninvasive measurement, and possibility of detection of multiple components in cultivation media, which in turn can provide tools for the quantification of more than one metabolite/substrate/product in real time. In this work, soft sensor based on excitation-emission fluorescence is for the first time applied for the monitoring of biotransformation production of 2-phenylethanol (2-PE) by yeast strains. Main process parameterssuch as optical density, glucose, and 2-PE concentrationswere determined with high accuracy and precision by fluorescence fingerprinting coupled with partial least squares regression. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:299-307, 2017