Abstract Xylose-rich solutions were produced by applying water pre-hydrolysis on mixed hardwood chips followed by an enzymatic treatment of the filtrate. The liquid phase obtained after water pre-hydrolysis contained xylose and xylans as the major components as well as other hemicelluloses and monosaccharides, acetic acid, and some degradation products of the monosaccharides [furfural, 5-(hydroxymethyl)furfural]. This hydrolysate underwent a two-step enzymatic treatment successively with endo-1,4-β-xylanase and 1,4-β-xylosidase to specifically depolymerize xylans into xylose without producing monosaccharides from the other oligosaccharides. After this treatment, 20.5% of the xylose units remained as oligomers. Matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry was applied after each step of the treatment to approach the structure of hemicellulose oligomers, to follow their depolymerization and to understand the reason for the resistance of some of them to enzymatic hydrolysis. It was found that highly acetylated xylans and/or xylans still substituted with 4-O-methylglucuronic groups were particularly resistant to this treatment.