Primary or secondary amino groups are widespread in biomolecules, being found in proteins and peptides as ε-amino groups of lysines and N-terminal amino groups. Additionally, numerous natural and synthetic small molecules contain amino groups, while amino-modified DNAs can be synthesized via solid-phase methods or enzymatic processes. These amino groups are amenable to modification through acylation using various activated carbonyl compounds. Of these, activated N-hydroxysuccinimide esters are particularly well-suited for applications in biolabeling and bioconjugation. In contrast to alternative activated esters, NHS derivatives exhibit excellent stability in aqueous environments. The ideal pH range for coupling amines with NHS esters in aqueous buffers is 8.3-8.5. Additionally, labeling can be performed in organic solvents, with polar aprotic solvents like DMF or DMSO being preferred. To ensure efficient amine acylation in non-aqueous solutions, the addition of one equivalent of tertiary amine base, such as triethylamine or diisopropylethylamine (DIPEA), is essential.