A 3D-QSAR model was generated for the prediction of anti-prion activity. The model was used in virtual screening protocol coupled with molecular docking on PrPC. Nine out of eleven selected compounds had a non-toxic profile and showed in vitro anti-prion activity. Compound 96 is a promising hit interfering with pathological transition of PrPC to PrPSc. 96 displays a fluorescence staining profile suggesting its application as a diagnostic for TSE.