Polymyxin B and E (colistin), are a group of cationic charged cyclic antibiotic lipopeptides that are frequently used in the clinics to treat infections caused by the multidrug-resistant gram-negative bacteria. Since the interactions with the blood plasma drug-transport proteins may play a critical role in determining their pharmacological and pharmacokinetic profiles, we studied the binding properties of polymyxins to the human serum albumin (HSA) under simulated physiological conditions by the combination of biophysical approaches, such as isothermal titration calorimetry (ITC), fluorescence anisotropy, circular dichroism (CD) buttressed by computational studies. The HSA binding to the polymyxins was relatively strong (K-a approximate to 1.0 x 10(7) M-1). Molecular docking indicated that polymyxins bind to the cleft of HSA between domains I and III via the electrostatic interactions. This evidence was further confirmed by the entropy-driven interaction for the polymyxins bound HSA. Far UV-CD experiments showed that the secondary structure of HSA doesn't alter and its stable structure is preserved. Collectively, these investigations revealed that the polymyxins bind preferentially to the partially unfolded intermediate forms of the protein structure; however, HSA molecule does not undergo any significant conformational changes upon binding. This is promising as it may limit the unfavorable side effects of the medicine. On the whole, the results provide quantitative and qualitative insight of the binding interaction between HSA and polymyxins, which is important in understanding their effect as therapeutic agents.