Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with beta-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of beta-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that) beta-galactosidase from Aspergillus oryzae has the highest affinity toward formation of beta-(1 -> 3) or beta-(1 -> 6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.