The article describes a novel bioorthogonal reaction, the cycloaddition of s-tetrazine and trans-cyclooctene derivatives, which proceeds at extremely fast rates without the need for catalysis. This reaction is highly versatile, tolerating a broad range of functionalities and occurring in high yield in various solvents, including organic solvents, water, cell media, and cell lysates. The rate constant for the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is particularly rapid (k2 2000 M−1s−1), enabling protein modification at low concentrations. The authors demonstrate the practicality of this method by successfully functionalizing thioredoxin (Trx) with a trans-cyclooctene derivative and subsequent ligation with s-tetrazine. The reaction is also shown to be compatible with biological functionalities, as evidenced by its efficiency in cell media and aqueous solutions containing rabbit reticulocyte lysate. The study highlights the potential of this fast bioconjugation method for assembling complex biomaterials under dilute conditions and for intracellular assembly of large molecular structures.The article describes a novel bioorthogonal reaction, the cycloaddition of s-tetrazine and trans-cyclooctene derivatives, which proceeds at extremely fast rates without the need for catalysis. This reaction is highly versatile, tolerating a broad range of functionalities and occurring in high yield in various solvents, including organic solvents, water, cell media, and cell lysates. The rate constant for the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is particularly rapid (k2 2000 M−1s−1), enabling protein modification at low concentrations. The authors demonstrate the practicality of this method by successfully functionalizing thioredoxin (Trx) with a trans-cyclooctene derivative and subsequent ligation with s-tetrazine. The reaction is also shown to be compatible with biological functionalities, as evidenced by its efficiency in cell media and aqueous solutions containing rabbit reticulocyte lysate. The study highlights the potential of this fast bioconjugation method for assembling complex biomaterials under dilute conditions and for intracellular assembly of large molecular structures.