Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in alpha-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining alpha-galactosidases. The fluorescently labeled aziridine-based probes 3 and 4 inhibit the two human retaining alpha-galactosidases alpha Gal A and alpha Gal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both alpha-galactosidases in cell extracts, thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells versus those derived from Fabry patients.