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The gut microbiome expresses a multitude of enzymes degrading polysaccharides in dietary plant fibers and in host-secreted mucus. The quantitative detection of these glycan-degrading enzymes in fecal samples is important to elucidate the functional activity of the microbiome in health and disease. We describe a protocol for detection of glycan-degrading enzyme activity in mouse and human fecal samples, namely sulfatase and four carbohydrate-active enzymes. Assessing their activity can inform treatment strategies for diseases linked to t