The copper-catalyzed azide-alkyne cycloaddition (CuAAC), a key example of click chemistry, has been widely applied in bioconjugation due to its high efficiency and selectivity. However, challenges arise in bioconjugation due to the sensitivity of biomolecules. This study presents an optimized CuAAC protocol for efficient bioconjugation, addressing issues such as oxygen sensitivity, catalyst stability, and byproduct formation. The optimized protocol uses a sulfonated bathophenanthroline ligand (THPTA) in a 5:1 ligand-to-copper ratio, which allows for efficient reaction under atmospheric oxygen conditions. Sodium ascorbate is used as the reducing agent, with a concentration of 5 mM to maintain active Cu(I) catalyst. The protocol also includes aminoguanidine to prevent oxidative damage to proteins by scavenging reactive oxygen species. The study demonstrates the effectiveness of the protocol in various bioconjugation applications, including the conjugation of siRNA with a fluorescent azide, the modification of RNA, and the coupling of proteins to virus-like particles. The protocol is robust and can be applied in a wide range of conditions, with the exception of substrates that may bind copper ions or have sterically hindered functional groups. Key considerations for successful CuAAC bioconjugation include maintaining the correct copper concentration (50–100 μM), using a suitable buffer (phosphate, carbonate, or HEPES), and avoiding the addition of ascorbate without the ligand. The protocol also suggests using alternative ligands or metal ions in cases where copper binding is problematic. Overall, the optimized CuAAC protocol provides a reliable and efficient method for bioconjugation, minimizing the risk of protein damage and ensuring high reaction yields. The study highlights the importance of careful optimization and the use of appropriate additives to achieve successful bioconjugation in various biological systems.The copper-catalyzed azide-alkyne cycloaddition (CuAAC), a key example of click chemistry, has been widely applied in bioconjugation due to its high efficiency and selectivity. However, challenges arise in bioconjugation due to the sensitivity of biomolecules. This study presents an optimized CuAAC protocol for efficient bioconjugation, addressing issues such as oxygen sensitivity, catalyst stability, and byproduct formation. The optimized protocol uses a sulfonated bathophenanthroline ligand (THPTA) in a 5:1 ligand-to-copper ratio, which allows for efficient reaction under atmospheric oxygen conditions. Sodium ascorbate is used as the reducing agent, with a concentration of 5 mM to maintain active Cu(I) catalyst. The protocol also includes aminoguanidine to prevent oxidative damage to proteins by scavenging reactive oxygen species. The study demonstrates the effectiveness of the protocol in various bioconjugation applications, including the conjugation of siRNA with a fluorescent azide, the modification of RNA, and the coupling of proteins to virus-like particles. The protocol is robust and can be applied in a wide range of conditions, with the exception of substrates that may bind copper ions or have sterically hindered functional groups. Key considerations for successful CuAAC bioconjugation include maintaining the correct copper concentration (50–100 μM), using a suitable buffer (phosphate, carbonate, or HEPES), and avoiding the addition of ascorbate without the ligand. The protocol also suggests using alternative ligands or metal ions in cases where copper binding is problematic. Overall, the optimized CuAAC protocol provides a reliable and efficient method for bioconjugation, minimizing the risk of protein damage and ensuring high reaction yields. The study highlights the importance of careful optimization and the use of appropriate additives to achieve successful bioconjugation in various biological systems.