Prebiotics are nondigestible substrates that stimulate the growth of beneficial microbes in the human intestine. Galactooligosaccharides (GOS) are food ingredients that possess prebiotic properties, in particular, promoting the growth of bifidobacteria in situ. However, precise mechanistic details of GOS consumption by bifidobacteria remain poorly understood. Because GOS are mixtures of polymers of different lengths and linkages, there is interest in determining which specific structures provide prebiotic effects to potentially create better supplements. This paper presents a method comprising porous graphitic carbon separation, isotopic labeling, and mass spectrometry analysis for the structure-specific analysis of GOS isomers and their bacterial consumption rate. Using this strategy, the differential bacterial consumption of GOS by the bifidobacteria species Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, and Bifidobacterium adolescentis was determined, indicating that the use of specific GOS isomers in infant formula may provide enrichment of distinct species.