Bioorthogonally modulated photoresponsive systems have been developed for applications in chemical biology. These photolabile protecting groups enable the controlled release of therapeutic agents by light, paving the way for photoactivated chemotherapy in which drug activation can be regulated with high spatial and temporal precision. Tetrazine-modified fluorogenic probes combining rotational and tetrazine-based quenching achieve selective fluorescence activation only when anchored to DNA-binding proteins in the nucleus. We demonstrated that both a bioorthogonal Diels–Alder reaction and light irradiation are necessary to release and activate drug payloads, confirming that bioorthogonally activatable photolabile systems represent a promising strategy for targeted drug release and selective imaging in live biological contexts.