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Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals., Light‐induced protein labeling: Cyclopropenone‐caged dibenzoannulated bicyclononynes (photo‐DMBO) are photo‐activatable dienophiles that engage in rapid inverse electron‐demand Diels–Alder cycloadditions with tetrazines upon light‐induced decarbonylation. Site‐specific incorporation of methyl‐tetrazine amino acids allows photo‐induced protein labeling in living cells with spatio‐temporal control using photo‐DMBO fluorophore conjugates.