The characterisation of the malaria aspartic protease flap loop dynamics upon open-flap inhibitor binding provided several conclusions. First, several alternative binding modes are observed for majority of 2aminoquinazolin-4(3H)-one based plm inhibitors. These binding modes are believed to be key transition states on the ligand binding pathway, and plays role in the ligand flap pocket substituent guiding under the flap loop and flap loop opening. Second, introduction of larger flap pocket substituent reduces the conformational space available by the ligand and shifts the binding preference to the binding mode observed in the crystal structure. This results in improved inhibitor activity, since role of the alternative binding modes is diminished. Third, poor 2-aminoquinazolin-4(3H)-one based inhibitor potency against plm V and catD is a result of ligands inability to bind under the flap loop. Binding modes identified suggest that the ligand binding under the flap loop in plm II is initiated by the hydrophobic interactions in-between the flap loop and adjacent α-helix; such interactions in plm V and catD are unfavourable. Taken together, our findings confirm that plm inhibitor selectivity can be achieved by targeting the flap loop with hydrophobic substituents that enables ligand binding under the flap loop, as such behaviour is not observed for several other aspartic proteases. The insight on the molecular determinants of ligand selectivity between plm II, plm V and catD provide a clear indication for the structure-based rational design of selective inhibitors. The ability to estimate compound selectivity before they are synthesized is of great importance in drug design, therefore, we expect that approach used here will be a valid support in selective inhibitor design not only against aspartic proteases, but other enzymes as well.