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This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in all compartments of Escherichia coli by Förster Resonance Energy Transfer (FRET) using the Superfolder mTurquoise2 ox-mNeonGreen FRET pair (sfTq2 ox -mNG). This FRET pair has more than twice the detection range for FRET interaction studies in the cytoplasm or periplasm of E. coli compared to other pairs to date. These protein-interaction studies can be performed in vivo because fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell. sfTq2 ox and mNG fluorescent protein fusions are co-expressed in bacterial cells and the fluorescence emission spectra are measured. By also measuring reference spectra for the background, sfTq2 ox -only and mNG-only samples, expected emission spectra can be calculated. Sensitized emission for mNG above the expected spectrum can be attributed to FRET and quantified by spectral unmixing. This bio-protocol discusses the sfTq2 ox -mNG FRET pair and provides a practical guide in preparing the protein fusions, setting up and running the FRET experiments, measuring fluorescence spectra and gives the tools to analyze the collected data., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)