As drug-binding kinetics has become an important factor to be considered in modern drug discovery, this work evaluated the ability of the Milestoning method in computing the absolute dissociation rate of a ligand from the serine–threonine kinase, glycogen synthase kinase 3β, which is a target for designing drugs to treat diseases such as neurodegenerative disorders and diabetes. We found that the Milestoning method gave good agreement with experiment with modest computational costs. Although the time scale for dissociation lasted tens of seconds, the collective molecular dynamics simulations total less than 1μs. Computing the committor function helped to identify the transition states (TSs), in which the ligand moved substantially away from the binding pocket. The glycine-rich loop with a serine residue attaching to its tips was found to undergo large movement from the bound to the TSs and might play a role in controlling drug-dissociation kinetics.