A simple and rapid dual-wavelength spectroscopic method is used for simultaneous determination of pentoses and hexoses in the prehydrolyzate from lignocellulosic biomass. The method is based on the following reaction mechanism: in the solution of hydrochloric acid, phloroglucinol gives color reaction with sugars or their degradation products, showing maximum absorbance at 553 nm and 410 nm. Based on dual-wavelength spectrophotometric measurement, the pentoses and hexoses can separately be quantified. It was found that the derivatives from these two different sugars have an isosbestic point at 425 nm. According to the validation results, high accuracy and reasonable recovery rate is shown with the present method (pentoses recovery 97.1 to 100.0%, hexoses recovery 97.2 to 102.0%). Additionally, the interferences from substances including lignin, furfural, 5-hydroxymethyl furfural (HMF), glucuronic acid, and galacturonic acid are insignificant. All of the above results illustrate the suitability of this method for analyzing sugars in the lignocelluloses prehydrolyzate, especially hardwoods or herbaceous plants, based on forest-related biorefinery research. INTRODUCTION Resource and energy shortages have become widespread issues for global development. As one of the most abundant renewable resources, lignocelluloses show great potential for future development, such as the production of transportation fuels and other value-added chemicals, while yielding lower greenhouse gas emissions. In the paper industry, lignocellulosic biomass is usually used for the production of chemical pulps, typically by alkaline pulping process. During the pulping process, a large amount of carbohydrates, mainly hemicelluloses, are released into the pulping liquor, which increases the loading in the spent liquor (black liquor) and provides few benefits (due to the lower heating value) to liquor combustion in the chemical recovery process. One of the key processes in the proposed "Integrated Forest Biorefinery" (van Heiningen 2006) is the pre-extraction of hemicelluloses for the production of fuel-based ethanol or biodiesel. In the prehydrolysis research, it is important to seek efficient methods and conditions for achieving a controlled sugar extraction yield (mainly from hemicelluloses) with minimum cellulose degradation. Data from the sugar content in the prehydrolyzate provides important information for better understanding the effects of the process PEER-REVIEWED ARTICLE bioresources.com Chi et al. (2013). "Sugar analysis of hydrolysates," BioResources 8(1), 172-181. 173 parameters, such as reaction temperature, time, acidity, alkalinity, etc., as they relate closely to the degree of hemicellulose hydrolysis and cellulose degradation. Many traditional methods are available for sugar analysis. Colorimetric methods, including the dinitrosalicylic acid (DNS) assay (Miller 1959), the orcinol-sulfuric acid method (Scott and Melvin 1953), and the phenol-sulfuric acid method (Dubois et al. 1956), can just be used for quantifying total reducin...