Bacteria need continuously to exchange small hydrophilic molecules with the exterior while keeping toxic substances out. In particular the sophisticated cell wall in Gram-negative bacteria presents a true barrier: made of two hydrophobic membranes separating a hydrophilic peptidoglycan layer. Among other factors there is a correlation between antibiotic resistance and modification in the outer cell wall composition, in particular changes in membrane channels called porins. To understand the molecular basis of antibiotic resistance caused by low cell wall permeability we need to quantify each component. However quantification of the membrane permeability is difficult caused by the absence of appropriated methods. Here we give a brief introduction on the current available techniques contributing to the knowledge of antibiotic permeation in Gram-negative bacteria.