The carbohydrate composition of lignocellulosic biomass hydrolysates is highly complex. High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), a widely used method for carbohydrate analysis, provides limited chemical information on the detected peaks. To improve the detection and increase the chemical information of the carbohydrates, HPAEC was coupled with mass spectrometry (MS). Using a pooled hydrolysate sample, it was shown that HPAEC-MS can separate and detect many oligosaccharides in one experimental run based on retention time and mass. The method was validated on its linearity, reproducibility and response factors. The analysis of a group of different biomass hydrolysates revealed that remaining disaccharides was the bottleneck of the hydrolysis process. As an analytical tool, HPAEC-MS provides information for the improvement of hydrolysate pretreatment method and enzyme cocktail quality. Besides, the consumption ability of microbial host strains for various mono- and oligosaccharides in hydrolysates can be assessed. (C) 2013 Elsevier Ltd. All rights reserved.