The quality of wine greatly depends on the features of the yeast used in its production, and yeast cell viability is one of the most important quality control issues to consider in this regard. In the first steps of winemaking, the use of a low-cost and simple methodology for monitoring the cell viability of yeast inoculates is of paramount importance. Gravitational field-flow fractionation is a useful technique for the determination of cell viability because it provides gentle experimental conditions, although the proper use of fluorophore probes as biomass indicators is required. In this paper the use of different fluorescent probes such as carboxyfluorescein diacetate (cFDA), calcein-AM, and SYTO-13 were considered as viability biomarkers. Calceina-AM allowed the establishment of a direct GrFFF method to determine cell viability, with a limit of detection of 5.0 x 10(4) viable cell/mL. SYTO-13 could be used as biomass indicator with a limit of detection of 3.5 x 10(4) total cells/mL. The suitability of the procedure was tested with three commercial yeast samples, and the results were compared with those obtained using standard techniques.