Three experimentally characterised inhibitors (1-3) and a fragment binder (4) with unknown functional effect were used to validate the methodology (Figure ). Our protocol successfully identified 1 as a potent allosteric inhibitor . 2, a fragment obtained by deconstructing inhibitor 1 that only shows very weak activity experimentally was classified as inactive by our protocol. 3, a covalently bound fragment that weakly inhibit PTP1B shows activity intermediate between 1 and 2 in our protocol . Fragment binder 4 is predicted functionally inactive by our approach. Additionally, we use various protocols to assess the effect of restraining a ligand orientation on the predicted activity levels of the protein. Through comparative analysis of the computed protein conformational ensembles we identify specific protein conformational states that could be used as blueprints for virtual screens of novel PTP1B allosteric modulators. Our efforts illustrate how our joint sMD/MSM protocol could be used to prioritise fragment hits for hit-to-lead chemistry efforts, and to plan virtual screening campaigns.