Aberrant Notch pathway function is associated with a wide array of developmental disorders, cancers, and neurodegenerative diseases. Thus, strategies to modulate Notch signaling may facilitate therapeutic intervention. Ligand binding to Notch receptors at the cell surface results in a series of cleavage events that release Notch intracellular domain (NICD) fragments that translocate to the nucleus where they function as transcriptional activators of downstream transcriptional programs. We have developed a cell-based assay that can be used to screen for modulators of NICD signaling by engineering HeLa cervical cancer cells with a Notch response element driving β-lactamase (BLA) reporter gene expression along with a tetracycline-inducible NICD expression system. Induction of NICD expression leads to increased BLA reporter activity that can be knocked down using NICD-specific RNA interference as well as RNA interference against endogenous components of the NICD transactivation complex. Profiling of 19 known compounds in this assay identified several previously undescribed modulators of NICD signaling. The Wnt pathway inhibitor ICG-001 antagonized NICD signaling, whereas the histone deacetylase inhibitor suberoylanilide hydroxamic acid, the heat shock protein 90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin, and the dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin inhibitor PI-103 each further activated the NICD-driven reporter activity. The AKT inhibitor triciribine and the PI3K inhibitor GDC-0941 also resulted in the enhanced reporter activity, strongly implicating a role for the PI3K/AKT pathway in regulating NICD signaling. Together this cell-based assay system provides a sensitive, quantitative readout for NICD signaling that is amenable to high-throughput screening for NICD pathway modulators.