This 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 derivatives Basic Protocol 2: Implementation of the sulfo-click reaction Basic Protocol 3: Synthesis of 3'-azido-4'-(carboxamido)ethane-sulfonyl azide-3'-deoxythymidine Basic Protocol 4: Detailed synthetic procedure for one-pot double-click conjugations.