Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Gal alpha(1,4)-GlcNAc and Man beta(1,4)-GlcNAc appendages. Mammalian glycosyl-transferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the beta-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural alpha-Gal and beta-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal beta-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal beta-GlcNAc moiety, by treatment with beta-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.