While softwoods represent a potential feedstock for second generation ethanol production, compounds present in their hydrolysates can inhibit fermentation. In this study, a novel Design of Experiments (DoE) approach was used to identify significant inhibitory effects on Saccharomyces cerevisiae D(5)A for the purpose of guiding kinetic model development. Although acetic acid, furfural and 5-hydroxymethyl furfural (HMF) were present at potentially inhibitory levels, initial factorial experiments only identified ethanol as a significant rate inhibitor. It was hypothesized that high ethanol levels masked the effects of other inhibitors, and a subsequent factorial design without ethanol found significant effects for all other compounds. When these non-ethanol effects were accounted for in the kinetic model, R-2 was significantly improved over an ethanol-inhibition only model ((R) over bar (2) = 0.80 vs. 0.76). In conclusion, when ethanol masking effects are removed, DoE is a valuable tool to identify significant non-ethanol inhibitors and guide kinetic model development. (C) 2014 Elsevier Ltd. All rights reserved.