The hydrophobic interaction between antimicrobial peptides and membrane hydrophobic cores is usually related to their cytotoxicity. In this study, the adsorption mechanism of five plasma membrane-associated peptides, indolicidin (IL) and its four derivatives, with hydrophobic ligands was investigated to understand the relationship between peptide hydrophobicity and bioactivity. The hydrophobic adsorption mechanisms of IL and its derivatives were interpreted thermodynamically and kinetically by reversed-phase chromatography (RPC) analysis and surface plasmon resonance (SPR) measurement, respectively. IL and its derivatives possess a similar random coil structure in both aqueous and organic solvents. Thermodynamic analysis showed that the binding enthalpy of peptides with higher electropositivity was lower than those with lower electropositivity and exhibited unfavorable binding entropy. Higher electropositivity peptides adsorbed to the hydrophobic surface arising from the less bound solvent on the peptide surface. A comparison with the kinetic analysis showed that IL and its derivatives adopt a two-state binding model (i.e., adsorption onto and self-association on the hydrophobic acyl chain) to associate with the hydrophobic surface, and the binding affinity of peptide self-association correlates well with peptide hemolysis. Consequently, this study provided a novel concept for understanding the action of plasma membrane-associated peptides.