4H-Pyrazoles are emerging as useful click reagents. Fluorinating the saturated center enables 4H-pyrazoles to react rapidly as Diels–Alder dienes without a catalyst but compromises the stability of these dienes under physiological conditions. To identify more stable 4H-pyrazoles for bioorthogonal chemistry applications, we investigated the Diels–Alder reactivity and biological stability of three 4-oxo-substituted 4H-pyrazoles. We found that these dienes undergo rapid Diels–Alder reactions with endo-bicyclo[6.1.0]non-4-yne (BCN) while being much more stable to biological nucleophiles than their fluorinated counterparts. We attribute the rapid Diels–Alder reactivity of the optimal oxygen-substituted pyrazole to a combination of antiaromaticity, predistortion, and spirocyclization. Their reactivity and stability suggest that 4-oxo-4H-pyrazoles can be useful bioorthogonal reagents.