The conventional equilibrium dialysis and ultrafiltration methods cannot be used to determine the protein binding of some peptides because of their non-specific adsorption on the semipermeable membrane or poor stability in the plasma. The method of dextran-coated charcoal adsorption combined with LC-MS/MS were used. Based on the kinetic principle of initial rate of candidate drugs absorbed to dextran-coated charcoal, seven phosphorylated peptides with the same amino acid sequence and different configurations in rat plasma were selected as the study model using; the protein binding in rat plasma were determined; the amino acid distribution rules affecting the changes in protein binding rates of peptide candidate drugs were summarized. The results suggest that the dextran charcoal adsorption method, as a supplementary method for the determination of plasma protein binding, is suitable for peptides or organic drug candidates that cannot be determined by traditional techniques.