Forster resonance energy transfer ( FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that ( 1) FRET spectra can be recorded in single bilayers, supported on a surface, and ( 2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.