A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo-giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics-cephalosporins and fluoroquinolones-through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.