We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (kFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. kFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. kFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, kFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The kFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.