Significance Human breast milk is rich in a family of structurally diverse unconjugated glycans. These human milk oligosaccharides (HMOs) can shape the intestinal microbiome, serve as soluble decoys for receptors of pathogens, and have immune-modulatory properties. Virtually nothing is known about the importance of the molecular complexity of HMOs for binding and biological activity, which hampers exploitation of their biomedical potential. We have developed a synthetic approach that can provide highly complex, asymmetrical, multiantennary HMOs. These compounds have been used in the development of a glycan microarray, which makes it possible to examine the biology of individual compounds. Binding studies with the array uncovered that the complex architecture of HMOs greatly affects protein–glycan binding.