The study of cellular glycosylation presents many challenges due, in large part, to the nontemplated nature of glycan biosynthesis and glycans' structural complexity. Chemoenzymatic glycan labeling (CeGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CeGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CeGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CeGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.