The force required to rupture the streptavidin-biotin complex was calculated using molecular simulations, showing good agreement with recent single-molecule AFM experiments. These simulations suggest a multi-step rupture mechanism involving five major unbinding steps. The binding force and specificity are attributed to a hydrogen bond network between biotin and streptavidin residues. Water bridges enhance complex stability and dominate binding interactions. Steric restraints do not contribute significantly to binding forces, though conformational motions were observed. Molecular recognition is essential for biological systems, but understanding binding and unbinding pathways and the molecular basis of specificity remains challenging. Experimental techniques like AFM provide insights into binding forces, while molecular dynamics (MD) simulations calculate binding free energies. However, these methods have limitations in capturing the actual unbinding pathway's force profile. To model the AFM experiment, extended MD simulations were used, pulling biotin out of the streptavidin binding pocket. The simulations revealed a detailed rupture mechanism, with the force profile showing multiple peaks corresponding to hydrogen bond and water bridge ruptures. The rupture process involved several steps, including the rupture of hydrogen bonds and water bridges, leading to the release of biotin. The simulations showed that the rupture force depends on pulling velocity, with a linear relationship at low velocities. The calculated rupture forces matched experimental data, suggesting a detailed mechanism involving hydrogen bonds and water bridges. The results highlight the role of hydrogen bonding and water bridges in stabilizing the complex and the complexity of the unbinding pathway. The study provides insights into the molecular mechanisms of streptavidin-biotin interactions and suggests further research into the energetics of the rupture process.The force required to rupture the streptavidin-biotin complex was calculated using molecular simulations, showing good agreement with recent single-molecule AFM experiments. These simulations suggest a multi-step rupture mechanism involving five major unbinding steps. The binding force and specificity are attributed to a hydrogen bond network between biotin and streptavidin residues. Water bridges enhance complex stability and dominate binding interactions. Steric restraints do not contribute significantly to binding forces, though conformational motions were observed. Molecular recognition is essential for biological systems, but understanding binding and unbinding pathways and the molecular basis of specificity remains challenging. Experimental techniques like AFM provide insights into binding forces, while molecular dynamics (MD) simulations calculate binding free energies. However, these methods have limitations in capturing the actual unbinding pathway's force profile. To model the AFM experiment, extended MD simulations were used, pulling biotin out of the streptavidin binding pocket. The simulations revealed a detailed rupture mechanism, with the force profile showing multiple peaks corresponding to hydrogen bond and water bridge ruptures. The rupture process involved several steps, including the rupture of hydrogen bonds and water bridges, leading to the release of biotin. The simulations showed that the rupture force depends on pulling velocity, with a linear relationship at low velocities. The calculated rupture forces matched experimental data, suggesting a detailed mechanism involving hydrogen bonds and water bridges. The results highlight the role of hydrogen bonding and water bridges in stabilizing the complex and the complexity of the unbinding pathway. The study provides insights into the molecular mechanisms of streptavidin-biotin interactions and suggests further research into the energetics of the rupture process.