AbstractThis article contains detailed synthetic procedures for the implementation of the sulfo‐click reaction to nucleoside derivatives. First, 3′‐O‐TBDMS‐protected nucleosides are converted to their corresponding 4′‐thioacid derivatives in three steps. Then, various conjugates are synthetized via a biocompatible and chemoselective coupling procedure using sulfonyl azide partners. Finally, to illustrate the potential of the sulfo‐click reaction, a nucleoside bearing two orthogonal azido groups is synthesized and engaged in one‐pot dual labeling through a sulfo‐click/copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) cascade. The high efficiency of the sulfo‐click reaction as applied to nucleosides opens up new possibilities in the context of bioconjugation. © 2020 Wiley Periodicals LLC.Basic Protocol 1: General protocol for the synthesis of 4′‐thioacid‐nucleoside derivativesBasic Protocol 2: Implementation of the sulfo‐click reactionBasic Protocol 3: Synthesis of 3′‐azido‐4′‐(carboxamido)ethane‐sulfonyl azide‐3′‐deoxythymidineBasic Protocol 4: Detailed synthetic procedure for one‐pot double‐click conjugations