The article presents a Cu-free variant of click chemistry that can label biomolecules such as glycans and lipids rapidly and selectively in living systems, overcoming the toxicity associated with traditional Cu-catalyzed reactions. The key reagent, a substituted cyclooctyne (DIFO), features ring strain and electron-withdrawing fluorine substituents, promoting a [3 + 2] dipolar cycloaddition with azides installed metabolically. This Cu-free click reaction has comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells without apparent toxicity. The authors demonstrate the technique's effectiveness by studying the dynamics of glycan trafficking in live cells, identifying a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics. The method offers a promising approach for dynamic imaging of biomolecules in living systems, particularly for glyobiology applications.The article presents a Cu-free variant of click chemistry that can label biomolecules such as glycans and lipids rapidly and selectively in living systems, overcoming the toxicity associated with traditional Cu-catalyzed reactions. The key reagent, a substituted cyclooctyne (DIFO), features ring strain and electron-withdrawing fluorine substituents, promoting a [3 + 2] dipolar cycloaddition with azides installed metabolically. This Cu-free click reaction has comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells without apparent toxicity. The authors demonstrate the technique's effectiveness by studying the dynamics of glycan trafficking in live cells, identifying a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics. The method offers a promising approach for dynamic imaging of biomolecules in living systems, particularly for glyobiology applications.