Antimicrobial resistance (AMR) is a global health threat of increasing concern, predicted to result in a ‘post-antibiotic’ era with ten million deaths per year by 2050 if new approaches are not developed. More research is needed into the discovery and development of new antibiotics to effectively treat infections. Just as importantly, we need new diagnostics that can rapidly identify bacterial infections, allowing for moretimely and appropriate therapy. Fluorescent derivatives of antibiotics have great potential to be applied to AMR research, from improving our basic understanding of antibiotic modes of action to the development of non-invasive imaging for clinical diagnostics. Better understanding how multidrug-resistant (MDR) bacteria can evade current and novel antibiotics requires a better understanding of the chemical biology of antibiotic action. This necessitates using new tools and techniques to advance our knowledge of bacterial responses to antibiotics, ideally in live cells in real time, to selectively investigate bacterial growth, division, metabolism, and resistance in response to antibiotic challenge. In this review, we discuss the preparation and biological evaluation of fluorescent antibiotics, focussing on how these reporters and assay methods can help elucidate resistance mechanisms. We also examine the potential utility of such probes for real-time in vivo diagnosis of infections. Better understanding how multidrug-resistant (MDR) bacteria can evade current and novel antibiotics requires a better understanding of the chemical biology of antibiotic action. This necessitates using new tools and techniques to advance our knowledge of bacterial responses to antibiotics, ideally in live cells in real time, to selectively investigate bacterial growth, division, metabolism, and resistance in response to antibiotic challenge. In this review, we discuss the preparation and biological evaluation of fluorescent antibiotics, focussing on how these reporters and assay methods can help elucidate resistance mechanisms. We also examine the potential utility of such probes for real-time in vivo diagnosis of infections. an enzyme produced by resistant bacteria that degrades β-lactam antibiotics by destroying the cyclic core. the core structure for a commonly used green fluorophore. a commonly used fluorophore core structure. a group of enzymes involved in DNA synthesis that are inhibited by the antibiotic trimethoprim. a blue-emitting fluorophore. enzymes produced by resistant bacteria capable of degrading most β-lactam antibiotics. widely used as a fluorescent biomarker. a commonly used measure of the efficacy of an antibiotic, measuring the lowest concentration that inhibits growth of a microorganism. bacteria that are resistant to multiple antibiotics and present the greatest threat to human health. a fluorophore emitting in a 700–1700-nm window that allows for tissue penetration and in vivo imaging. involved in cell wall peptidoglycan synthesis and the target of β-lactam antibiotics. a common whole-body imaging technique that can generate 3D images based on detecting pairs of gamma rays produced by a positron-emitting radioisotope attached to a biologically active molecule that binds to specific targets within the organism. a common whole-body imaging technique that can generate 3D images by measuring gamma rays produced by a radioisotope, usually attached to a biologically active molecule that binds to specific targets within the organism. a medicinal chemistry investigation of the effects of changes in chemical structure on biological activity.