N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT) has previously been shown to effectively block the production of amyloid β (Aβ) in cultured cells and transgenic animals. In the present study, DAPT analogues were synthesized and evaluated for the prevention of the proteolytic processing of amyloid precursor protein (APP) as γ-secretase inhibitors by using a highly efficient cell-based reporter gene assay for γ-secretase. This study revealed that the γ-secretase inhibition could be improved with analogous benzodiazepine bearing C-5 cyclohexyl substituent with maintenance of N-terminal difluorophenylacetyl group as compared to DAPT. Our results also indicated the stringent binding site for the alanyl residue of DAPT whose modification with one carbon variation dramatically diminished its inhibitory effect on γ-secretase.