In this presentation I will discuss work focused on the major research theme of the Laboratory of Chemical Glycobiology, which is centered on the design and use of chemical biology tools to probe and perturb glycans and carbohydrate processing enzymes in cells and in vivo. Topics to be discussed will include our studies on the function of the enzymes that regulate levels of the intracellular O-GlcNAc modification, the creation and characterization of inhibitors of these enzymes, and how these have led to industrial efforts that have advanced such compounds into human clinical trials. I will also describe recent work on the creation and optimization of live cell fluorescence imaging agents that allow quantitative measurement of the activity of glycosidases in live mammalian cells by microscopy or flow cytometry. Examples on the use of these discovery-focused chemical probes to advance fundamental research will be presented. These examples will include studies on examining the roles of O-GlcNAc in nutrient sensing and cellular proteostasis, as well as developing high throughput screening methods to identify modulators of carbohydrate processing enzymes implicated in various diseases ranging from cancer to neurodegeneration.