The article discusses the optimization and application of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction for bioconjugation. The authors highlight the challenges and limitations of the CuAAC reaction in biological contexts, such as the sensitivity to oxygen and the potential for protein damage or precipitation. They describe a refined protocol that overcomes these issues by using sodium ascorbate as a reducing agent, optimizing the ligand/Cu ratio, and employing aminoguanidine to mitigate the toxicity of ascorbate byproducts. The protocol is demonstrated through successful bioconjugation experiments with siRNA, bacteriophage Qβ, and bovine serum albumin (BSA), showing high efficiency and selectivity. The key elements for successful bioconjugation include the use of sodium ascorbate, maintaining Cu concentrations between 50 and 100 μM, using at least five equivalents of the tris(heterocycle)methylamine ligand, and avoiding certain substrates that may strongly bind copper ions or have sterically hindered azide or alkyne groups.The article discusses the optimization and application of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction for bioconjugation. The authors highlight the challenges and limitations of the CuAAC reaction in biological contexts, such as the sensitivity to oxygen and the potential for protein damage or precipitation. They describe a refined protocol that overcomes these issues by using sodium ascorbate as a reducing agent, optimizing the ligand/Cu ratio, and employing aminoguanidine to mitigate the toxicity of ascorbate byproducts. The protocol is demonstrated through successful bioconjugation experiments with siRNA, bacteriophage Qβ, and bovine serum albumin (BSA), showing high efficiency and selectivity. The key elements for successful bioconjugation include the use of sodium ascorbate, maintaining Cu concentrations between 50 and 100 μM, using at least five equivalents of the tris(heterocycle)methylamine ligand, and avoiding certain substrates that may strongly bind copper ions or have sterically hindered azide or alkyne groups.