Mucin-type O-linked glycosylation is a fundamental post-translational modification that is involved in a variety of important biological processes. However, the lack of chemical tools to study mucin-type O-linked glycosylation has hindered our molecular understanding of O-linked glycans in many biological contexts. The review discusses the significance of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases in biology and development of chemical tools to study these enzymes and their substrates.