Gaining an in-depth understanding of the response of Saccharomyces cerevisiae to the different inhibitors generated during the pretreatment of lignocellulosic material is driving the development of new strains with higher inhibitor tolerances. The objective of this study is to assess, using flow cytometry, how three common inhibitors (vanillin, furfural, and acetic acid) affect the membrane potential, the membrane permeability and the concentration of reactive oxygen species (ROS) during the different fermentations. The membrane potential decreased during the detoxification phase and reflected on the different mechanisms of the toxicity of the inhibitors. While vanillin and furfural caused a metabolic inhibition and a gradual depolarization, acetic acid toxicity was related to fast acidification of the cytosol, causing an immediate depolarization. In the absence of acetic acid, ethanol increased membrane permeability, indicating a possible acquired tolerance to ethanol due to an adaptive response to acetic acid. The intracellular ROS concentration also increased in the presence of the inhibitors, indicating oxidative stress. Measuring these features with flow cytometry allows a real-time assessment of the stress of a cell culture, which can be used in the development of new yeast strains and to design new propagation strategies to pre-adapt the cell cultures to the inhibitors.