γ-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer’s disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. γ-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the γ-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of ~3,000 compounds. The overall assay performance was robust due to a calculated Z′ factor of 0.74 and its demonstrated ability to identify known γ-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of γ-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.