Gene regulatory proteins, such as transcription factors (TFs), bind to DNA and orchestrate spatial and temporal gene expression patterns. This regulation relies on dynamic and coordinated protein-protein interactions, either through direct pairwise interactions or as part of multiprotein complexes. Therefore, the development of methods to assay these interactions within the cellular context is crucial. Förster resonance energy transfer combined with fluorescence lifetime imaging microscopy (FRET-FLIM) is a powerful quantitative imaging technique for detecting protein-protein interactions in vivo. This approach involves labeling the proteins of interest with fluorescent tags and measuring changes in the fluorescence lifetime of the donor fluorophore. The reduction in donor lifetime in the presence of the acceptor fluorophore provides direct evidence of a physical interaction between the proteins under study. In this chapter, we present a detailed and simple protocol for the acquisition and analysis of FRET-FLIM data using the Leica STELLARIS 8 FALCON FLIM Microscope® system, illustrated with two interacting transcription factor proteins as an example.