Polymyxin B remains an antibiotic of last resort because of its toxicities. Although newer therapies are becoming available, it is anticipated that resistance to these agents will continue to emerge, and understanding the safest and most efficacious manner to deliver polymyxin B will remain highly important. Recent data have demonstrated that polymyxin B may be less nephrotoxic than colistin. Pharmacokinetically, polymyxin B is primarily eliminated via non-renal pathways, and most do not recommend adjusting the dose for renal impairment. However, some recent studies suggest a weak relationship between polymyxin B clearance and patient creatinine clearance. This review article will describe the clinical pharmacokinetics of polymyxin B and address relevant issues in chemistry and assays available.