Breast milk, the sole source of nourishment for newborns, has been under intense selective pressure over millions of years of evolution to meet the infant’s needs to grow and survive. Interestingly, the third most abundant component class in human milk, oligosaccharides, is both non-nutritive and non-digestible. The oligosaccharides play a key role in creating and maintaining a healthy infant gut microbiota through two established mechanisms: prebiotic effects and competition for specific pathogen. The functions of milk glycans (oligosaccharides, glycoproteins and glycolipids) are related to their specific structures. However, identifying and quantifying those structures has been a major challenge in their analysis due to the complexity of the structures. Therefore, functional studies to date have involved either a limited number of structures or little or no structural information. Novel mass spectrometry-based glycomic tools allowed to differentiate and quantitate not only glycans of different mass but also those with the same masses but different sugar composition and linkages. The advances in glycomic analysis have allowed us to observe the correlation between the consumption of individual glycan structures and gut bacteria population. Animal studies which require sufficient amounts of the material will further reveal how the complex fluid nourishes infants and protects them from disease in human body. This has necessitated research directed towards development of methods for large-scale separation of the biomolecules. Bovine milk is a potentially excellent source of commercially viable analogs to human milk glycansn. We recently isolated and discovered that whey permeate, a secondary product from cheese production, contains oligosaccharides structurally far more similar to human milk oligosaccharides than currently available prebiotics. The combination of advanced analytics with the new engineering capabilities allowed for high molecular resolution and separation techniques that can be scaled- up to semi-industrial and industrial scale for translation of lab-based discoveries.