In order to remove or reduce the concentrations of toxic substances present in the sugarcane bagasse hemicellulose hydrolysate for xylose-to-xylitol bioconversion, the hydrolysate was pretreated by changing the initial pH level through the combination of different bases and acids with or without the subsequent addition of activated charcoal. Attention was given to the influence of the fermentation time as well.The experiments were based on multivariate statistical concepts, with the application of fractional factorial design techniques to identify the important variables in the process. Subsequently, the levels of these variables were quantified by the response surface methodology, which permitted the establishment of the best pretreatment procedure with a xylose-to-xylitol bioconversion efficiency of 86.2%. This procedure consisted in increasing the pH of the hydrolysate from 0.5 to 7.0 with CaO and reducing it to 5.5 with H3PO4. Next, the hydrolysate was treated with activated charcoal (2.4%). The highest xylitol yield (0.79 g/g) corresponded to a productivity of 0.47 g/L/h.