Fluorescence Resonance Energy Transfer (FRET), the non-radiative transfer of energy from an excited fluorophor (donor) to a suitable acceptor (quencher). The transfer is influenced by the distance, the dipolar orientation, and the spectral overlap of the donor and acceptor moieties. In FRET substrates, fluorophor and quencher are linked tightly by a peptide chain. Enzymatic cleavage is accompanied by increasing fluorescence, as donor and acceptor are separated.