Fermentation by microorganisms remains to be a critical step for optimization in the conversion of biomass to ethanol to make the process economical.   It is well known that pretreatment of feedstocks generates hydrolysates containing toxic compounds (acetate, furfural, HMF, etc), which can severely inhibit the growth and fermentation performance of microorganisms.  Although a great deal of research has been conducted to evaluate the ethanologen tolerance to the inhibitors in hydrolysates, this work has rarely provided insights into the mechanisms of toxicity.  Using the ethanologen, Zymomomas mobilis 8b as a model organism, we present tools for examining hydrolysate toxicity using flow cytometry and fluorescence microscopy analyses of cells stained with dyes such as Syto 9 and propidium iodide grown in the presence of specific inhibitory compounds as well as hydrolysate.    Development of these tools can lead to research for improving the cell viability under the stress of hydrolysates which will ultimately enhance the fermentation performance of an organism.