As the second most common polysaccharides in nature, hemicellulose has received much attention in recent years for its importance in biomass conversion in terms of producing high yields of fermentable sugars and value-added products, as well as its role in reducing biomass recalcitrance. Therefore, a time and labor efficient method that specifically analyzes hemicellulose content would be valuable to facilitate the screening of biomass feedstocks. In this study, a one-step acid hydrolysis method was developed, which applied 4 wt% sulfuric acid at 121°C for 1 h to rapidly quantify XGM (xylan + galactan + mannan) contents in various types of lignocellulosic biomass and model hemicelluloses. This method gave statistically identical results in XGM contents compared to results from conventional two-step acid hydrolysis while significantly shortening analysis time. Biotechnol. Bioeng. 2014;111: 1088–1096. © 2014 Wiley Periodicals, Inc.