Overcoming the effects of hydrolysate toxicity towards ethanologens is a key technical barrier in the biochemical conversion process for biomass feedstocks to ethanol. Despite its importance, the complexity of the hydrolysate toxicity phenomena and the lack of systematic studies, analysis and tools surrounding this issue have blocked a full understanding of relationships involving toxic compounds in hydrolysates and their effects on ethanologen growth and fermentation. In this study, we developed a quantitative, high-throughput biological growth assay using an automated turbidometer to obtain detailed inhibitory kinetics for individual compounds present in lignocellulosic biomass hydrolysate. Information about prolonged lag time and final cell densities can also be obtained. The effects of furfural, hydroxymethylfurfural (HMF), acetate and ethanol on growth rate and final cell densities of Zymomonas mobilis 8b on glucose are presented. This method was also shown to be of value in toxicity studies of hydrolysate itself, despite the highly colored nature of this material. Using this approach, we can generate comprehensive inhibitory profiles with many individual compounds and develop models that predict and examine toxic effects in the complex mixture of hydrolysates, leading to the development of improved pretreatment and conditioning processes as well as fermentation organisms.