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Fluorescent proteins (FPs) are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non-native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging and Förster resonance energy transfer (FRET). To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise2ox (sfTq2 ox ) and is able to withstand challenging, oxidizing environments. sfTq2 ox has improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide bonds. The improved sfTq2 ox has the same spectroscopic properties as mTq2, that is, high fluorescence lifetime and quantum yield. The sfTq2 ox -mNeongreen FRET pair allows the detection of periplasmic protein-protein interactions with energy transfer rates exceeding 40%. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way for in vivo antibiotic screening. SIGNIFICANCE: The periplasmic space of Gram-negative bacteria contains many regulatory, transport and cell wall-maintaining proteins. A preferred method to investigate these proteins in vivo is by the detection of fluorescent protein fusions. This is challenging since most fluorescent proteins do not fluoresce in the oxidative environment of the periplasm. We assayed popular fluorescent proteins for periplasmic functionality and describe key factors responsible for periplasmic fluorescence. Using this knowledge, we engineered superfolder mTurquoise2ox (sfTq2 ox ), a new cyan fluorescent protein, capable of bright fluorescence in the periplasm. We show that our improvements come without a trade-off from its parent mTurquoise2. Employing sfTq2 ox as FRET donor, we show the direct in vivo interaction and disruption of unique periplasmic antibiotic targets FtsB and FtsL., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)