The article discusses the development and application of two bioorthogonal reactions: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition (Cu-free click chemistry). Bioorthogonal reactions are designed to be inert to biological systems, selectively reactive under biocompatible conditions, and nontoxic to cells and organisms. The Staudinger ligation, based on the classic reduction of azides with triarylp phosphines, forms an amide bond and has been successfully used in live mice. However, it has slow kinetics and the phosphine reagents are cytotoxic. The strain-promoted azide-alkyne cycloaddition, inspired by the rapid reaction of cyclooctyne with phenyl azide, is a "click" reaction that is less cytotoxic. The authors optimized cyclooctyne reagents by modifying their structure to enhance reactivity and stability, achieving high sensitivity and efficiency in labeling azide-modified biomolecules in live cells and organisms. These reactions have enabled precise chemical modification of biomolecules and real-time visualization of molecules and processes in living systems, contributing to significant advancements in biological discovery and biotechnology.The article discusses the development and application of two bioorthogonal reactions: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition (Cu-free click chemistry). Bioorthogonal reactions are designed to be inert to biological systems, selectively reactive under biocompatible conditions, and nontoxic to cells and organisms. The Staudinger ligation, based on the classic reduction of azides with triarylp phosphines, forms an amide bond and has been successfully used in live mice. However, it has slow kinetics and the phosphine reagents are cytotoxic. The strain-promoted azide-alkyne cycloaddition, inspired by the rapid reaction of cyclooctyne with phenyl azide, is a "click" reaction that is less cytotoxic. The authors optimized cyclooctyne reagents by modifying their structure to enhance reactivity and stability, achieving high sensitivity and efficiency in labeling azide-modified biomolecules in live cells and organisms. These reactions have enabled precise chemical modification of biomolecules and real-time visualization of molecules and processes in living systems, contributing to significant advancements in biological discovery and biotechnology.