Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are char- acterized by low permeability and often contain drug e ux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or e ux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identi ed clear correlations between e ux activ- ity and drug di usion and accumulation in both susceptible and resistant strains, and have also clari ed how certain chemical structures can a ect drug entry and residence time within the cell. In this Perspective, we discuss the biological underpinnings of drug permeability and export using several prototypical in ux and e ux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid e orts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics.