Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.