). Secondly, a dissociation analysis must be performed for at least one representative compound, to check if it dissociates in a single step. We favor τRAMD, as it provides an enhanced sampling of the dissociation pathway without defining collective variables. But alternative protocols could be equally valid. If those two conditions are met, we expect a high confidence in the activity cliff predictions via their differences in structural stability. As confirmation, we performed a prospective validation with the described steps. Due to recent insights into structural stability pointing at crystallized ligands having higher structural stability, we tested activity cliff prediction on a new system exclusively on ligands with a crystal structure. We used BACE1 as a test system, a pharmacologically relevant target with a less defined key interaction than the hinge interaction in kinases. From the set of 50 ligands from the PDBbind, forming 82 pairs, the proportion of cliffs to ligands was lower than in CDK2 due to the high scaffold diversity within the set. The location, number, and size of the changes have a strong impact (i.e., a series where all substitutions occur at the same substitution site will be able to form more cliffs than if they were dispersed, and the same is true for the size and number of substitutions). Despite the structural diversity, the main interacting groups were maintained across all ligands: an amide nitrogen interacts with the backbone oxygen of G291. Thus, we hypothesized that the dissociation mechanism is shared across the series, and the conclusions extracted with the reference compound should apply to the whole BACE1 dataset.