Bacterial infections are increasingly difficult to treat due to the worldwide increase in antibiotic resistance. The development of new antibiotics is notoriously difficult and expensive. The majority of research is based on either in vitro target inhibition assays or in vivo random screening assays. In the former the developed antibiotics fail because they cannot pass the bacterial envelope or are promiscuous and in the latter the antibiotics often fail because they are toxic to eukaryotes. Target specific assays in living bacteria are much more cost effective because the chance of toxicity is lower as the target has been chosen carefully and positive compounds can pass the envelope. Recently, it is appreciated that the penicillin binding proteins that synthesize the peptidoglycan layer collaborate with and are activated by other proteins. These protein complexes are interesting novel targets for antibiotic development. Unfortunately, no assays exist that measure the interaction of these proteins in the periplasm. By developing a Fluorescence Energy Transfer (FRET) assay in the periplasm, like we did for the cytoplasm, we fill this omission in the toolbox. In addition, the FRET assay will be the first assay that is able to measure proteins interactions in the periplasm in living cells. The obtained information on localization and interactions as a function of the bacterial division cycle will be, in combination with the data available for the cytoplasm, used to build a computer model on the molecular mechanisms of bacterial envelope growth that can be used to identify antibiotic targets.