SummaryThe bacterial cell division machinery is organized in the so‐called divisome composed of highly dynamic but low abundant interacting (membrane‐bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background‐insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ‐ring and the so‐called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan‐synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto‐ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto‐ring elements.