Bioorthogonal reactions are chemical reactions that do not interfere with biological systems. These reactions involve functional groups that are inert to biological components, selectively react under biocompatible conditions, and are non-toxic to cells and organisms. They are particularly useful for in vivo applications, where they can modify biomolecules without disrupting their natural functions. The development of bioorthogonal reactions has been guided by a need to understand functional group and reactivity spaces outside those defined by nature, with a focus on stability, kinetics, and selectivity. The Staudinger ligation and strain-promoted azide–alkyne cycloaddition (also known as "Cu-free click chemistry") are two notable bioorthogonal reactions that have been developed in the laboratory. The Staudinger ligation uses azides and phosphines to form amide bonds, while the strain-promoted azide–alkyne cycloaddition uses cyclooctynes and azides to form triazole products. Both reactions have been tested in various biological environments, including cultured cells and live organisms. The Staudinger ligation has been used to label cell surface glycans in live mice, while the strain-promoted azide–alkyne cycloaddition has been used to label azide-modified biomolecules in cultured cells and in live organisms such as Caenorhabditis elegans, zebrafish, and mice. These reactions have enabled the high-precision chemical modification of biomolecules in vitro and the real-time visualization of molecules and processes in cells and live organisms. The field of bioorthogonal chemistry is now a well-established area of reaction methodology and an important tool for biological discovery. The development of these reactions has been driven by the need to understand the molecular interactions and chemical transformations that enable life, and to develop new tools for studying biological processes. The success of bioorthogonal chemistry has been facilitated by the availability of commercial kits that provide azide or alkyne-labeled biomolecules and complementary probes for detection or enrichment. These kits have enabled the use of bioorthogonal chemistry by non-experts, which is essential for widespread adoption of the technology outside of chemistry circles. The future of bioorthogonal chemistry lies in the continued development of new reactions and the optimization of existing ones to achieve the best balance of reactivity and stability. The field has also benefited from the contributions of physical organic chemists, who have provided insights into the mechanisms and theoretical basis of these reactions. The importance of reaction discovery as the foundation of bioorthogonal chemistry cannot be overstated, as the few prototype reactions that underpin current bioorthogonal transformations were discovered long before the chemistry/biology interface became a popular research area. The success of bioorthogonal chemistry is a testament to the power of interdisciplinary collaboration and the importance of fundamental research in advancing the field.Bioorthogonal reactions are chemical reactions that do not interfere with biological systems. These reactions involve functional groups that are inert to biological components, selectively react under biocompatible conditions, and are non-toxic to cells and organisms. They are particularly useful for in vivo applications, where they can modify biomolecules without disrupting their natural functions. The development of bioorthogonal reactions has been guided by a need to understand functional group and reactivity spaces outside those defined by nature, with a focus on stability, kinetics, and selectivity. The Staudinger ligation and strain-promoted azide–alkyne cycloaddition (also known as "Cu-free click chemistry") are two notable bioorthogonal reactions that have been developed in the laboratory. The Staudinger ligation uses azides and phosphines to form amide bonds, while the strain-promoted azide–alkyne cycloaddition uses cyclooctynes and azides to form triazole products. Both reactions have been tested in various biological environments, including cultured cells and live organisms. The Staudinger ligation has been used to label cell surface glycans in live mice, while the strain-promoted azide–alkyne cycloaddition has been used to label azide-modified biomolecules in cultured cells and in live organisms such as Caenorhabditis elegans, zebrafish, and mice. These reactions have enabled the high-precision chemical modification of biomolecules in vitro and the real-time visualization of molecules and processes in cells and live organisms. The field of bioorthogonal chemistry is now a well-established area of reaction methodology and an important tool for biological discovery. The development of these reactions has been driven by the need to understand the molecular interactions and chemical transformations that enable life, and to develop new tools for studying biological processes. The success of bioorthogonal chemistry has been facilitated by the availability of commercial kits that provide azide or alkyne-labeled biomolecules and complementary probes for detection or enrichment. These kits have enabled the use of bioorthogonal chemistry by non-experts, which is essential for widespread adoption of the technology outside of chemistry circles. The future of bioorthogonal chemistry lies in the continued development of new reactions and the optimization of existing ones to achieve the best balance of reactivity and stability. The field has also benefited from the contributions of physical organic chemists, who have provided insights into the mechanisms and theoretical basis of these reactions. The importance of reaction discovery as the foundation of bioorthogonal chemistry cannot be overstated, as the few prototype reactions that underpin current bioorthogonal transformations were discovered long before the chemistry/biology interface became a popular research area. The success of bioorthogonal chemistry is a testament to the power of interdisciplinary collaboration and the importance of fundamental research in advancing the field.