Antibiotic resistance is presenting an urgent global medical need. In particular for Gram-negative KAPE ‘superbugs’, no new antibiotics will be available for many years to come. ‘Old’ polymyxins are increasingly being used as the last-line therapy against KAPE pathogens which are resistant to all other antibiotics. Contemporary pharmacological data indicate that currently recommended dosage regimens of polymyxins are suboptimal and may lead to the increasing emergence of resistance reported in many countries. Resistance to polymyxins often implies a total lack of antibiotics for treatment of life-threatening infections caused by Gram-negative ‘superbugs’. Polymyxins exert their antibacterial effect by binding to lipid A in the bacterial outer membrane via specific polar and hydrophobic interactions, and subsequently causing membrane disruption and cell death. The most common mechanism of polymyxin resistance involves modifications of the phosphate groups of lipid A that prevent the interactions with polymyxins. Using our new structure-activity relationship model, we have designed a series of novel polymyxin analogues to target polymyxin resistance by increasing the ‘hydrophobic reach’ of the amino residues at position 6 and/or 7. These lipopeptides were prepared through a total synthesis approach. MICs, static time-kill and SEM/TEM results clearly demonstrated the antibacterial effects of our novel hits against polymyxin-resistant Pseudomonas aeruginosa. The pharmacokinetics of these hits were also examined in rats. Our findings suggest their potential as novel antibiotics against Gram-negative ‘superbugs’.