To understand the binding mechanism between designed peptides and thrombin, molecular dynamics simulation was carried out. In this study, the designed peptide binder in docking complex with human thrombin, and human thrombin carried its native binder (2-[2-(4-chloro-phenylsulfanyl)-acetylamino]-3-(4-guanidino-phenyl)-propionamide) were used for MD simulation. The docking complex was independently subjected to MD simulation using Gromacs-2020,[28 ] and the simulation system was filled with SPC/E water in a cubic box. The system was neutralized using Na+ and Cl−, and the distance between the edge of water box and docking complex was 15 Å. The system undergoes energy minimization using the steepest descent method, followed by equilibration using the isochoric–isothermal ensemble and isothermal–isovolumetric ensemble under 300 K for 100 and 200 ps, respectively. The production of MD simulation was carried out for 100 ns under 300 K, and we used the last 10 ns of simulation for gmxMMPBSA[29 (link)] analysis.