One of the major causes of antibiotic resistance in the Gram-negative bacteria is the low permeability across the outer membrane. Currently a main bottleneck in the development of effective antibiotics is the lack of a general method to quantify permeation which would allow screening for optimal scaffolds. Here, we present a permeation assay based on conventional electrophysiology. The method mainly involves application of concentration gradients of charged molecules with different electrophoretic mobilities through a membrane channel. Thus the unbalanced flux creates an electrostatic potential which provides direct information on relative ion fluxes. The experimental approach applied here involves measuring zero-current-potentials and the corresponding single channel conductance. For OmpC and the beta-lactamase inhibitor avibactam at a 10 gm gradient the calculated flux rate at V-m = 0mV was about n = 200 molecules/s per OmpC single pore. (C) 2017 Elsevier Inc. All rights reserved.