The intestinal glycome contains carbohydrates that govern the composition and metabolism of the gut microbiome. Gut microbes differentially access intestinal glycans as a nutrient source thereby directing microbial abundance and metabolite synthesis. Thus, identifying microbial accessible carbohydrates (MACs) in the gut is critical to understanding microbial ecologies and developing novel prebiotics that manipulate gut microbial populations for beneficial outcomes. However, no methods exist to efficiently examine MACs in biologically-derived mixtures. Here, we present a high-throughput platform to examine MACs from plant, animal, and microbial sources by simultaneously measuring bioluminescence from a genome-wide library of engineered Bacteroides thetaiotaomicron strains that harness bacterial glycan detection machinery. We demonstrate that this platform exhibits specific and sensitive responses to glycan mixtures and characterize a previously unknown MAC present in Saccharomyces cerevisiae. Expanding this technology across gut Bacteroides species will yield a diverse and broadly applicable glycomics platform to identify novel prebiotic substrates.