Template assistance allows organic reactions to occur under highly diluted conditions – where intermolecular reactions often fail to proceed – by bringing reactants into close spatial proximity. This strategy has been elegantly applied to numerous systems, but always with the retention of at least one of the templating groups in the product. In this report, we describe a traceless, templated amide-forming ligation that proceeds at low micromolar concentration under aqueous conditions in the presence of biomolecules. We utilized the unique features of an acylboronate–hydroxylamine ligation, in which covalent bonds are broken in each of the reactants as the new amide bond is formed. By using streptavidin as a template and acylboronates and O–acylhydroxylamines bearing desthiobiotins that are cleaved upon amide formation, we demonstrate that traceless, templated ligation occurs rapidly even at sub-micromolar concentrations. The requirement for a close spatial orientation of the functional groups – achieved upon binding to streptavidin – is critical for the observed enhancement in the rate and quantity of product formed.