Abstract High throughput screening (HTS) techniques are required for the fast hit inhibitors selection in the early discovery process. However, in Beta-secretase (BACE1) inhibitors screening campaign, the most frequently used methoxycoumarin based peptide substrate (M-2420) is not widely applicable when aromatic or heterocycle compounds of natural source show auto-fluorescence interferences. Here, in order to overcome these drawbacks, we propose the use of a highly selective 4-(4-dimethylaminophenylazo)benzoic acid/5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (DABCYL/1,5-EDANS) based peptide substrate (Substrate IV), whose cleavage product is devoid of spectroscopic interference. HrBACE1-IMER was prepared and characterized in terms of units of immobilised hrBACE1. BACE1 catalyzed Substrate IV cleavage was on-line kinetically characterized in terms of KM and vmax, in a classical Michaelis and Menten study. The on-line kinetic constants were found consistent with those obtained with the in solution fluorescence resonance energy transfer (FRET) standard method. In order to further validate the use of Substrate IV for inhibition studies, the inhibitory potency of the well-known BACE1 peptide InhibitorIV (IC50: 0.19±0.02μM) and of the natural compound Uleine (IC50: 0.57±0.05) were determined in the optimized on-line hrBACE1-IMER. The IC50 values on the hrBACE1-IMER system were found in agreement with that obtained by the conventional methods confirming the applicability of Substrate IV for on-line BACE1 kinetic and inhibition studies.