This study used MD simulations in combination with τRAMD to elucidate, for the first time, the mechanism of dissociation of agonists and antagonists of TLR8. Two unbinding pathways were identified, the external and internal paths. Surprisingly, some ligands showed clear preferences for one specific unbinding path in our simulations. The agonist only dissociated through the external path, while the cationic antagonists only dissociated through the internal path. Neutral antagonists were able to dissociate through both the internal and external paths. These differences in path