Hemicellulose fractions derived from lignocellulosic biomass acid pretreatments have been considered promising substrates for H-2 production via anaerobic fermentation. In this context, the objective of this study is to develop a high-performance liquid chromatography (HPLC) methodology for the simultaneous determination of glucose, xylose, arabinose, acetic acid, butyric acid, 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (furfural) in the complex matrices used for the H-2 production from hemicellulose fractions. HPLC analyses were performed using two columns under different conditions: the Aminex HPX-87H column (mobile phase consisted of 0.005 mol L-1 H2SO4 solution at a flow rate of 0.7 mL min(-1) and temperature of 60 degrees c) and the Rezex RFQ Fast acid (mobile phase consisted of 0.005 mol L-1 H2SO4 solution at a flow rate of 1.0 mL min(-1) and temperature of 80 degrees C). Acids (acetic and butyric acids) and inhibitors (HMF and furfural) were analyzed by UV-Vis detection at 210 nm, and C6 and C5-sugars (glucose, xylose and arabinose) were analyzed using a refractive index detector (RI). Some validation parameters, such as selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and robustness were successfully evaluated. The monitoring of fermentative media for H-2 production from different hemicellulose fractions was successfully performed using the proposed methodologies. The acid-pretreated samples from corn cob, cocoa husk, sugarcane straw and bagasse substrates presented a range of xylose contents from 84.85 to 222.98 mmol L-1, which resulted in a maximum production of 2.99 mol H-2/mol total carbohydrate. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.