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This paper addresses some key issues related to the automation of fermentation process analysis in the context of industrial-scale ethanol production from sugarcane substrates. As the current methods for the determination of cell density and viability are time consuming and laborious, high resolution in situ microscopy (0.5µm) is proposed as a promising alternative. Laboratory-scale experiments presented here show that this imaging technique allows automatic, on-line, and real-time monitoring of yeast cells suspended in sugarcane molasses used in the ethanol industry. In particular, the feasibility of cell concentration measurements of Saccharomyces cerevisiae SA-1 in industrial sugarcane molasses is demonstrated. Automated concentration measurements exhibit a linear correlation with manual reference values using a Neubauer chamber from 3×10 6 cells/mL up to a saturation level at approximately 2×10 8 cells/mL. Furthermore, it was demonstrated that the microscopic resolution of this technique, combined with its large statistics, allows a morphological assessment of the size, shape and some internal structures of the yeast cells. On average, the accuracy of the algorithm´s yeast cells classification was 0.80. The results obtained suggest that the ISM is a suitable tool to perform in-line sugarcane fermentation monitoring.