A copper-free click chemistry method has been developed for dynamic in vivo imaging of biomolecules. This approach allows rapid and selective labeling of glycans and lipids in living systems, overcoming the toxicity of traditional copper-catalyzed reactions. The key reagent is a substituted cyclooctyne, which promotes a [3 + 2] dipolar cycloaddition with azides metabolically installed into biomolecules. This reaction is as fast and non-toxic as the copper-catalyzed version, enabling the study of glycan trafficking and internalization dynamics in live cells. The method uses bioorthogonal reactions, where the cell's metabolic machinery installs azides into target biomolecules, which are then covalently labeled with a probe. The azide is a widely used reporter due to its small size, metabolic stability, and lack of reactivity with natural biofunctionality. However, existing azide-specific reactions, such as the Staudinger ligation and Cu-catalyzed azide–alkyne cycloaddition, have limitations in sensitivity and toxicity. The new copper-free click reaction combines the biocompatibility of the Staudinger ligation with the fast kinetics of click chemistry, offering high sensitivity and selectivity. The study demonstrates the ability to label and image cell-surface glycans in live cells, revealing dynamic trafficking and internalization processes. The method was tested in live cells and in a mouse model, showing no toxicity and enabling real-time monitoring of glycan dynamics. This approach has potential applications in glycobiology and other areas requiring real-time imaging of biomolecules in living systems.A copper-free click chemistry method has been developed for dynamic in vivo imaging of biomolecules. This approach allows rapid and selective labeling of glycans and lipids in living systems, overcoming the toxicity of traditional copper-catalyzed reactions. The key reagent is a substituted cyclooctyne, which promotes a [3 + 2] dipolar cycloaddition with azides metabolically installed into biomolecules. This reaction is as fast and non-toxic as the copper-catalyzed version, enabling the study of glycan trafficking and internalization dynamics in live cells. The method uses bioorthogonal reactions, where the cell's metabolic machinery installs azides into target biomolecules, which are then covalently labeled with a probe. The azide is a widely used reporter due to its small size, metabolic stability, and lack of reactivity with natural biofunctionality. However, existing azide-specific reactions, such as the Staudinger ligation and Cu-catalyzed azide–alkyne cycloaddition, have limitations in sensitivity and toxicity. The new copper-free click reaction combines the biocompatibility of the Staudinger ligation with the fast kinetics of click chemistry, offering high sensitivity and selectivity. The study demonstrates the ability to label and image cell-surface glycans in live cells, revealing dynamic trafficking and internalization processes. The method was tested in live cells and in a mouse model, showing no toxicity and enabling real-time monitoring of glycan dynamics. This approach has potential applications in glycobiology and other areas requiring real-time imaging of biomolecules in living systems.