Unique carbohydrate antigens are expressed on the surface of various pathogens, including bacteria, parasites, and viruses, and aberrant glycosylation is a frequent feature of cancer cells. Antibodies recognizing such carbohydrate antigens may be used for the specific detection of potentially harmful cells, immunohistochemistry, and diagnostic and therapeutic applications. The generation of specific and strongly binding antibodies against defined carbohydrate epitopes is challenging, since isolated carbohydrates often suffer from low purity, usually have limited immunogenicity, and induce antibodies of low affinity. We describe a protocol to generate highly affine monoclonal antibodies (mAbs) against pure and defined synthetic carbohydrate antigens. First, an oligosaccharide is covalently coupled to an immunogenic carrier protein to obtain a glycoconjugate. This glycoconjugate is used to raise oligosaccharide-specific antibodies in mice, and splenocytes are fused with myeloma cells to form hybridomas. Hybridoma clones producing oligosaccharide-specific mAbs are selected by glycan microarray screening. Selected clones are expanded and mAbs are purified from the cell culture supernatant. This protocol is suitable to procure carbohydrate-specific mAbs of high specificity, selectivity, and affinity that may be useful for a variety of biochemical and medical applications.