The interactions between intestinal mucins and the gut microbiota significantly influence digestive processes, metabolic activities, and pathogen resistance in the gastrointestinal tract. To explore this dynamic relationship, this chapter describes a workflow for studying the degradation of mucin oligosaccharides from isolated mucins. The approach involves purifying mucins from intestinal tissues and then subjecting them to a mixture of fecal glycosidases originating from commensal bacteria. The remaining oligosaccharides attached to the mucins after glycosidase treatment are released and then characterized and quantified using liquid chromatography-mass spectrometry (LC-MS)2. This approach identifies relevant intestinal oligosaccharides that are degraded by commensal flora. By combining natural oligosaccharide characterization with measurement of their degradation, we expand the repertoire of glycan structures for studying the intestinal glycomic degradome. Few commercially available characterized glycans or glycoproteins exist that cover the unique type of glycan mixtures found in the intestines where most microbes in the gastrointestinal tract reside. The presented workflow uses relevant glyco-substrates to investigate mucus degradation patterns, which are crucial for discerning pathological conditions associated with intestinal dysbiosis.