NFQ1, a non-fluorescent quencher, is used for a new type of One Sample Fluorescence Resonance Energy Transfer (OS-FRET) method employing a novel, non-fluorescent methanethiosulfonate-linked acceptor that can be reversibly coupled to a target sulfhydryl residue via a disulfide bond. After the quenched donor emission is quantitated the acceptor is removed by reduction, enabling measurement of unquenched donor emission in the same sample.