The pharmacokinetics of polymyxin B1, polymyxin B2, colistin A, and colistin B were investigated in a rat model following intravenous administration (0.8 mg/kg) of each individual component. Plasma and urine concentrations were determined by LC-MS/MS, and plasma protein binding was measured by ultracentrifugation. Total and unbound pharmacokinetic parameters for each component were calculated using noncompartmental analysis. All of the polymyxin components had a similar clearance, volume of distribution, elimination half-life, and urinary recovery. The area under the concentration-time curve for polymyxins B1 and B2 was greater than those of colistins A and B. Colistin A (56.6 ± 9.25%) and colistin B (41.7 ± 12.4%) displayed lower plasma protein binding in rat plasma compared to polymyxin B1 (82.3 ± 4.30%) and polymyxin B2 (68.4 ± 3.50%). These differences in plasma protein binding potentially equate to significant differences in unbound pharmacokinetics, highlighting the need for more stringent standardization of the composition of commercial products currently available for clinical use.