The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-alpha (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J >= 5.1 x 10(13) M-1 cm(-1) nm(4)) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J >= 3.7 x 10(15) M-1 cm(-1) nm(4)) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.