AbstractTetrazine‐ and sydnone‐based click reactions have emerged as important bioconjugation strategies with fast kinetics and N2 or CO2 as the only byproduct. Mechanistic studies of these reactions have focused on the initial rate‐determining cycloaddition steps. The subsequent N2 or CO2 release from the bicyclic intermediates has been approached mainly through computational studies, which have predicted lifetimes of femtoseconds. In the present study, bioorthogonal cycloadditions involving N2 or CO2 extrusion have been examined experimentally at the single‐molecule level by using a protein nanoreactor. At the resolution of this approach, the reactions appeared to occur in a single step, which places an upper limit on the lifetimes of the intermediates of about 80 μs, which is consistent with the computational work.