Bioorthogonal chemistry enables the selective modification of biomolecules in complex biological systems. This review discusses the development and applications of bioorthogonal reactions, which are essential for studying biomolecules in their native environments. Key to these reactions is the ability to react rapidly and selectively under physiological conditions, avoiding interference with biological processes. The review covers the historical development of bioorthogonal reactions, starting with protein bioconjugation, and discusses the design of unique amino acid sequences to create orthogonal functionality for selective protein modification. It also explores the development of bioorthogonal transformations involving unnatural functional groups and methods to incorporate these groups into biomolecules. The review concludes with a discussion of new bioorthogonal chemical reactions and their applications to unexplored biological processes. Classic methods for protein modification include reactions with cysteine and lysine residues, such as disulfide exchange, alkylation, and Michael addition. Modern methods involve metal-mediated transformations and the N terminus as a target for modification. These methods include reductive alkylation, thiol-ene chemistry, and the use of transition metals for tyrosine and tryptophan modification. The N terminus can be modified through transamination reactions, which have been successfully applied for selective modification of proteins. Native chemical ligation (NCL) is a powerful method for protein modification, allowing the ligation of two highly functionalized molecules under physiological conditions. NCL has been used in conjunction with expressed protein ligation (EPL) and protein-trans splicing (PTS) to extend its applications to living systems. Unique amino acid sequences have been used to create new functionalities for selective chemical or enzymatic modification. Fluorogenic biarsenical and bisboronic acid reagents have been developed for labeling proteins, with applications in studying protein interactions and dynamics. Peptide tags detected through chelation of transition metals have been used for protein purification and labeling. Enzymatic modification of peptide tags, such as biotin ligase and lipoic acid ligase, has been used to selectively modify proteins in live cells. The aldehyde tag has been used to modify proteins expressed in E. coli and mammalian cells. Site-specific protein modification has also been accomplished using bacterial sortases and phosphopantetheinyl transferases. Bioorthogonal reactions, such as the condensation of ketones/aldehydes with aminooxy and hydrazide probes, the Staudinger ligation of azides and triarylphosphines, and the reactions of azides and alkynes, have been developed for the labeling of biomolecules. These reactions are essential for studying biomolecules in their native environments and have been applied to various biomolecule classes. The Staudinger ligation has been adapted for applications beyond biomolecule labeling, including protein synthesis. The copper-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) has become a widely used clickBioorthogonal chemistry enables the selective modification of biomolecules in complex biological systems. This review discusses the development and applications of bioorthogonal reactions, which are essential for studying biomolecules in their native environments. Key to these reactions is the ability to react rapidly and selectively under physiological conditions, avoiding interference with biological processes. The review covers the historical development of bioorthogonal reactions, starting with protein bioconjugation, and discusses the design of unique amino acid sequences to create orthogonal functionality for selective protein modification. It also explores the development of bioorthogonal transformations involving unnatural functional groups and methods to incorporate these groups into biomolecules. The review concludes with a discussion of new bioorthogonal chemical reactions and their applications to unexplored biological processes. Classic methods for protein modification include reactions with cysteine and lysine residues, such as disulfide exchange, alkylation, and Michael addition. Modern methods involve metal-mediated transformations and the N terminus as a target for modification. These methods include reductive alkylation, thiol-ene chemistry, and the use of transition metals for tyrosine and tryptophan modification. The N terminus can be modified through transamination reactions, which have been successfully applied for selective modification of proteins. Native chemical ligation (NCL) is a powerful method for protein modification, allowing the ligation of two highly functionalized molecules under physiological conditions. NCL has been used in conjunction with expressed protein ligation (EPL) and protein-trans splicing (PTS) to extend its applications to living systems. Unique amino acid sequences have been used to create new functionalities for selective chemical or enzymatic modification. Fluorogenic biarsenical and bisboronic acid reagents have been developed for labeling proteins, with applications in studying protein interactions and dynamics. Peptide tags detected through chelation of transition metals have been used for protein purification and labeling. Enzymatic modification of peptide tags, such as biotin ligase and lipoic acid ligase, has been used to selectively modify proteins in live cells. The aldehyde tag has been used to modify proteins expressed in E. coli and mammalian cells. Site-specific protein modification has also been accomplished using bacterial sortases and phosphopantetheinyl transferases. Bioorthogonal reactions, such as the condensation of ketones/aldehydes with aminooxy and hydrazide probes, the Staudinger ligation of azides and triarylphosphines, and the reactions of azides and alkynes, have been developed for the labeling of biomolecules. These reactions are essential for studying biomolecules in their native environments and have been applied to various biomolecule classes. The Staudinger ligation has been adapted for applications beyond biomolecule labeling, including protein synthesis. The copper-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) has become a widely used click