Bioorthogonal reactions are selective transformations that are not affected by any biological functional group and are widely used for chemical modification of biomolecules. Recently, we reported that vinylboronic acids (VBAs) gave exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels–Alder (iEDDA) reaction with tetrazines bearing a boron-coordinating pyridyl moiety compared to tetrazines lacking such a substituent. In this integrated experimental and theoretical study, we show how the reaction rate of the VBA-tetrazine ligation can be accelerated by shifting the equilibrium from boronic acid to the boronate anion in the reaction mixture. Quantum chemical activation strain analyses reveal that this rate enhancement is a direct consequence of the excellent electron-donating capability of the boronate anion in which the π HOMO is pushed to a higher energy due to the net negative potential of this species. We have explored the second-order rate constants of several tetrazines containing potential VBA-coordinating hydroxyl substituents. We observed an increase in rate constants of several orders of magnitude compared to the tetrazines lacking a hydroxyl substituent. Furthermore, we find the hydroxyl-substituted tetrazines to be more selective toward VBAs than toward the commonly used bioorthogonal reactant norbornene, and more stable in aqueous environment than the previously studied tetrazines containing a pyridyl substituent.