Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor-and acceptor-centric FRET efficiency (E-D and E-A) images of a single cell for obtaining the peak values (E-Di and E-m), thus yielding the corresponding stoichiometric ratios (E-Di/E-Ai) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and BcI-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria. (C) 2019 Elsevier Inc. All rights reserved.