Updated project metadata. A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added and removed on diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects elude methods to track O-GlcNAc response functions with chemical strategies: spatial control over subcellular locations and time control during labeling reactions. The objective of this study was to create intracellular proximity labeling tools to identify functional changes of O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on TurboID system for rapid protein biotin conjugation that was directed to O-GlcNAc protein modifications inside cells, a set of tools we call “GlycoID.” Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, label O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin as well as serum stimulation revealed functional changes in O-GlcNAc proteins in as soon as 30 minutes of signaling. We demonstrate that the GlycoID strategy can capture O-GlcNAcylated “activity hubs” consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events like insulin stimulation poises these tools to be used for determining mechanisms of glycobiological cell regulation. Our datasets in HeLa cells will be a useful functional resource for O-GlcNAc-associated physiology.