Dynamic interactions between RNA and RNA-binding proteins (RBPs) regulate a broad spectrum of bacterial functions. Here, we have characterised how the RBPome is dynamically rewired during the E. coli life cycle. We applied Orthogonal Organic Phase Separation (OOPS) coupled with an RNase digestion step to shortlist bona fide bacterial RBPs and assessed whether proteins bound RNA differentially express at different cell proliferation stages.