How to effectively delivering therapeutic agents, including gamma-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notchl intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of gamma-secretase inhibitor, DAFT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAFT alone could not block the gamma-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notchl, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hesl). Our data suggest that Notchl/Hes1 signaling pathway is deactivated using DAFT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notchl could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver gamma-secretase inhibitor DAFT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases. (C) 2017 Elsevier Inc. All rights reserved.