This thesis examined the mechanism of nephrotoxicity of the last-resort polymyxins using a systems approach. This study employed multiple complementary methods to investigate the accumulation of polymyxins in the kidneys and renal tubular cells, and identified a previously undiscovered mechanism by which polymyxins could cause nephrotoxicity. These discoveries will allow us to find innovative approaches to overcome polymyxin-induced kidney toxicity in patients, thereby optimising the dosage regimens. This study targets a significant global medical challenge, antibiotic resistance.