A hemicellulosic hydrolysate was prepared with 0.3 M H2SO4 at 98 °C for 1 h. The total initial reducing sugar was maintained at 45 g l−1 by synthetic xylose supplementation. The seven detoxification methods were employed including either the single addition of solid CaO (to pH 10 or 6) or its combinations with zeolite shaking. Over-liming gave the hydrolysate that was most completely fermented by Pichia stipitis and Candida shehatae at 30 °C, pH 6, among the tested methods.