Antibiotic resistance in Gram-negative pathogenic bacteria has reached alarming levels and these drug-resistant bacteria pose an urgent global threat. All of the major classes of antibiotics with activity against Gram-negative bacteria, including penicillins, cephalosporins, carbapenems, aminoglycosides, quinolones, and tetracyclines, have over time succumbed to widespread resistance. Fortunately, medicinal chemistry has been very successful at iteratively addressing the emergence of resistance (and other limitations) with second and third (or more) generation products. The polymyxin class has broad Gram-negative antimicrobial activity, including against bacteria resistant to the antibiotic classes listed above. It is because of such activity that polymyxins have witnessed a resurgence in clinical use, but toxicity is concerning and commonly limits dosing. And, unlike other classes of antibiotics, the polymyxin class has been recalcitrant to clinically meaningful optimization and only the initially discovered, natural polymyxins are available for clinical use. This article explores recent developments in polymyxin-based drug discovery and development and the extensive effort invested in trying to identify clinically viable second-generation variants.