β-Secretase 1 (BACE1) is the enzyme involved in the abnormal production of the amyloidogenic peptide Aβ42, one of the major causes of histological hallmarks of Alzheimer's disease. Thus, BACE1 represents a key target protein in the development of new potential drugs for the non-symptomatic treatment of Alzheimer's disease. Since the discovery of BACE1 one decade ago, both in the pharmaceutical industry and in academia there has been an intense search for novel inhibitors to be developed as new effective drugs. There is a great deal of interest in the discovery of selective non-peptide BACE1 inhibitors with a new chemical skeleton, suited for central nervous system penetration and endowed with more appropriate pharmacokinetic properties. Therefore, the selection of appropriate methods for screening and characterization of BACE1 inhibitors is crucial. This review focuses on the description of the in vitro methods to test BACE1 activity and inhibition, with particular emphasis on fluorescence resonance energy transfer (FRET) methods, aiming at critically highlighting advantages and drawbacks. An overview of BACE1 inhibitors is given, underlying the variability of the FRET methods reported in the literature, and the structure evolution of inhibitors active in cellular cultures and in vivo, from peptide to small synthetic and natural structures.