This article covers recent developments in the design and application of activity-based probes (ABPs) for glycosidases, with emphasis on the different enzymes involved in metabolism of glucosylceramide in humans. Described are the various catalytic reaction mechanisms employed by inverting and retaining glycosidases. An understanding of catalysis at the molecular level has stimulated the design of different types of ABPs for glycosidases. Such compounds range from (1) transition-state mimics tagged with reactive moieties, which associate with the target active site-forming covalent bonds in a relatively nonspecific manner in or near the catalytic pocket-to (2) enzyme substrates that exploit the catalytic mechanism of retaining glycosidase targets to release a highly reactive species within the active site of the enzyme, to (3) probes based on mechanism-based, covalent, and irreversible glycosidase inhibitors. Some applications in biochemical and biological research of the activity-based glycosidase probes are discussed, including specific quantitative visualization of active enzyme molecules in vitro and in vivo, and as strategies for unambiguously identifying catalytic residues in glycosidases in vitro.