Overcoming the effects of hydrolysate toxicity after pretreatment and/or enzymatic hydrolysis is a key technical barrier in the biochemical conversion process for biomass feedstocks to ethanol.   Yet, the complexity of the hydrolysate toxicity phenomena and the lack of systematic studies and tools surrounding this issue has prevented us from fully understanding relationships involving toxic compounds in hydrolysates, their relative inhibitory effects on ethanologen growth and fermentation, and the impact of various conditioning approaches to effectively mitigate these inhibitory effects.  We conducted systematic studies to analyze chemical composition of the hydrolysates and developed quantitative, high throughput biological growth assays to obtain the inhibitory kinetics for individual compounds, along with correlation of growth and fermentation performance in conditioned diluted acid corn stover hydrolysates and hydrolysate fractions.  These key findings provide important insights for understanding hydrolysate toxicity and provide guidance for potential process development in both pretreatment and hydrolysate conditioning operations, along with potential future strain improvement and tolerance strategies.  The tools that have been developed can also be more broadly applied to other feedstock and pretreatment process situations as well as other ethanologens.