The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2–10 nm. Spectral spillover-corrected quantitative (three-filterset) FRET is a cost-effective and versatile approach, which can be applied in flow cytometry and various modalities of fluorescence microscopy, where it provides pixel-by-pixel quantitative FRET efficiency values as well as FRET-corrected fluorescent intensities corresponding to the expression level of the two labeled interacting molecular species. Here, we provide a protocol for implementing such measurements, starting from the preparation of appropriate samples including controls, through a step-by step guide for image processing and derivation of microscopic FRET maps, to various aspects of interpreting the results. The image processing described is aided by a freely available ImageJ/Fiji plugin RiFRET which has been developed with the biology-focused users in mind. The interface guides the user through the evaluation process in a friendly manner and provides the option for automatic processing of large image libraries once the various calibration factors have been correctly set.