Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation. Protein channels in the outer membrane are known to form an entry route for many antibiotics into bacterial cells. Up until today, there has been a lack of simple experimental techniques to measure the antibiotic uptake and the local concentration in subcellular compartments. Hence, rules for translocation directly into the various Gram-negative bacteria via the outer membrane or via channels have remained elusive, hindering the design of new or the improvement of existing antibiotics. In this review, we will discuss the recent progress, both experimentally as well as computationally, in understanding the structure-function relationship of outer-membrane channels of Gram-negative pathogens, mainly focusing on the transport of antibiotics.