The focus of this study is to develop a process for conditioning spent liquor produced by SO2-ethanol water (SEW) fractionation of spruce chips/softwood biomass for fermentation to butanol, ethanol, and acetone/2-propanol (so-called ABE process) by Clostridia bacteria. This study is an integral part of a project which aims at creating an economic process that can utilize cheap forestry residues such as twigs, cones, treetops, branches, and bark to produce renewable chemicals and liquid fuels. The results of this study suggest that the proposed scheme for conditioning of SEW spent liquor from spruce/softwood biomass can be successfully applied to produce chemicals and biofuels by ABE fermentation. Butanol, acetone, and ethanol are produced at a total yield of around 0.25 g/g sugars.