Brewer's spent grain (BSG) samples were subjected to a two-step aqueous processing (starch extraction and autohydrolysis) in order to assess their potential as a raw material for obtaining a mixture of arabinoxylooligosaccharides (AXOS) suitable to be use as prebiotics for elderly. After hydrothermal treatment, the liquors were refined by a sequence of purification and conditioning steps including membrane filtration, enzymatic hydrolysis, and ion exchange. The presence of both substituted (degree of polimerization (DP) = 2-10) and unsubstituted (DP = 2-16) oligosaccharides made up of xylose and arabinose (AXOS) were confirmed in purified mixtures (in which total OS content = 84% w/w) by using chromatographic techniques and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Finally, AXOS were evaluated for their prebiotic activity by in vitro fermentation assays using fecal inocula from elderly people, demonstrating that AXOS were slightly better substrates than FOS, in terms of bacterial population shifts as in the production of SCFA.