The prehydrolysate stream from a Kraft dissolving pulp mill can be valorized by fermentation of the hemicellulosic sugars into biofuels or bioproducts, such as ethanol or butanol, instead of the typical practice of combustion to produce energy. An obstacle facing the use of Kraft hemicelluloses prehydrolysate for biofuels production is the low sugar concentration and the presence of fermentation inhibitors that include organic acids, furans and phenolic compounds. A precondition to ensure the survival of the fermentation microorganisms and to have high fermentation yields is to remove the inhibitors. Concentration of the prehydrolysate is also necessary to reduce the size of the processing equipment and decrease the energy cost. The purpose of this study was to develop a strategy for the concentration and detoxification of hemicelluloses prehydrolysate prior to its conversion into biofuels. Experiments were conducted to screen and select suitable organic membranes among 7 samples of reverse osmosis, nanofiltration, and ultrafiltration membranes. Three membranes (Dow NF270, Trisep TS40, and Trisep XN45) showed the highest sugar retentions relative to inhibitors removal. They were however not efficient for the removal of the phenolic compounds. It was also found that flocculation with ferric sulfate as coagulant could be utilized as a secondary detoxification step that can be combined with nanofiltration. The optimization of the flocculation step with a jar test showed that the highest phenolics removal (similar to 80%) can be obtained when the ratio of ferric ions to phenols is 1 g/g, and the pH is between 6.5 and 7.5. A new process concept for the detoxification and concentration has been developed based on these experimental results.