The clinical utility of the β-lactam antibiotics is threatened by resistant bacterial pathogens that produce β-lactamases. These enzymes hydrolytically degrade β-lactams, rendering them ineffective. Although several β-lactamase inhibitors have been approved for clinical use, currently used inhibitors only target a subset of the clinically relevant β-lactamases. Additionally, β-lactamase variants that exhibit reduced susceptibility to these inhibitors are increasing in prevalence. Thus, drug discovery efforts must continue to identify and develop novel β-lactamase inhibitors. Herein, we describe a luminescent biosensor, based on the β-lactam-inducible AmpR regulon, which can be used to quantify β-lactamase inhibition in bacterial cells. This approach does not require the use of expensive substrates and can account for factors such as outer membrane permeability, prioritizing the discovery of inhibitors that are effective against bacterial cells.