The identification of biomass pretreatment-generated compounds that impede cellulose hydrolysis is critical for improving the overall biomass saccharification process. The aim of this study was to correlate the identification and concentrations of switchgrass dilute acid pretreatment-generated compounds to cellulolytic enzyme inhibition and to tie this back to processing parameters. Preparation of 24 dilute acid prehydrolyzates was performed with switchgrass at temperatures from 140 to 180 °C, processing times from 10 to 40 min, and sulfuric acid concentrations of 0.5% or 1% (v/v). Results showed that all the switchgrass prehydrolyzates significantly reduced cellulolytic enzyme activities when assayed against model substrates. Exoglucanase was the most sensitive with its activity reduction, ranging from 58% to 88%; the inhibitory effect on β-glucosidase and the cellulase cocktail ranged from 32% to 63% and 16% to 41%, respectively. Polyphenolic compounds were the most detrimental pretreatment-generated pr...