The processing of lignocellulosic biomass to prepare it for conversion of the sugars present to ethanol is a critical step in the overall process.  Over the years, an increasingly detailed analysis of the resulting hydrolysate has demonstrated the complexity of this material.  Early on, the analysis of sugars present provided insights into metabolic engineering for increased ethanol yields resulting in the development of strains with the ability to convert both C5 and C6 sugars.  More recently, identification of a variety of potentially inhibitory compounds has sparked efforts to develop more robust strains through adaptation, selection, and pathway engineering.  Yet, it has become clear that these approaches are unlikely to lead to the development of a truly robust ethanologen because 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 and their relative inhibitory effects on ethanologen growth and fermentation.  We established a program to analyze the chemical composition of hydrolysate and hydrolysate fractions and developed quantitative, high throughput biological growth assays to obtain the inhibitory kinetics for individual compounds and mixtures, correlating those data with fermentation performance. These key findings provide important insights for understanding hydrolysate toxicity and provide guidance for metabolic engineering strategies directed toward strain improvement.