To determine the potential biofuel yield and necessary technological parameters a known concentration and type of fermentable sugars should be produced during chemical or biological extraction from lignocellulose. The most popular method for sugar interpretation and quantification is liquid chromatography (HPLC) using refractive index (RI) detector. The aim of this research was to show the applicability of the high–performance liquid chromatography using refractive index (HPLC RI) technique for sugar interpretation in hydrolysed hay and possible solutions for optimisation of this method. Analysis of hydrolysed hay with standard additive showed low recovery of sugar concentrations and inconsistencies with dinitrosalicylic acid (DNS) method, which was mostly due to low separation of peaks of these sugars on the chromatograms. As result HPLC RI method was useful for qualitative analysis of sugars only, not for its quantification.