A new approach to selective photodynamic therapy (PDT) was developed by designing chlorin e6 (Ce6)-containing macromolecules, which are sensitive to tumor-associated proteases. The agents are nontoxic in their native state but become fluorescent and produce singlet oxygen on protease conversion. Coupled with optimized delivery systems, we show that (a) the agents efficiently accumulate in tumors due to the enhanced permeability and retention effect, (b) the agents are locally activated by proteases, (c) local drug concentrations can be measured by quantitative fluorescence tomography, and (d) light-treated tumors show reduced growth. A single low dose of PDT (0.125 mg Ce6 equivalent/kg) was sufficient to suppress tumor growth by >50%. Activatable singlet oxygen generation agents provide increased efficacy with reduced toxicity, and it could become a powerful PDT.