Abstract Chemotactic-driven cell migration is an important biological process impacting many areas of life science research including immunology, cancer and developmental biology. The dominant high-throughput solution for measuring cellular chemotaxis in vitro is the Boyden-chamber. However, traditional Boyden chamber products have inherent liabilities including: a) the inability to directly visualize the cell migration process, b) a requirement of tens of thousands of cells per well, c) the need to label cells for quantitation, d) a relative insensitivity to surface integrin signaling, and e) high random migration artifacts. Microfluidic solutions have sought to eliminate some of these deficiencies, specifically by providing the ability to visualize the cell migration process, but at the expense of relatively low throughput and cumbersome quantitation methods. Using a live-cell imaging approach and real-time image analysis quantitation, we have developed a new technology which eliminates most of the deficiencies of existing Boyden chambers and most importantly, provides direct visualization of both migration and invasion as it occurs. Integrated metrics precisely quantify the chemotactic response using significantly fewer cells (500-5000 cells per well) in a 96-well format. We exemplify this novel image based technique by studying the migration and invasion of both non-tumor and tumor-derived cell lines. Using Fetal bovine serum (FBS) as a chemoattractant, HT-1080 and NIH-3T3 cells were evaluated for their ability to directionally migrate across a coated surface or invade through a basement membrane extract by embedding the cells in an extracellular matrix prior to seeding. Qualitatively, we observed significant morphological differences between migrating and invading cells, the latter adopting a mesenchymal morphology reminiscent of cells that have undergone epithelial-mesenchymal transition (EMT) and exhibiting thinner, spindle-like morphology as they invade through the matrix. Using both cell count and cell area metrics, we observed a significant difference between migration and invasion kinetics of the relatively non-invasive cell line, NIH-3T3, and the highly invasive human fibrosarcoma derived cell line, HT-1080. Moreover, we observed significant and selective inhibition of HT-1080 cell invasion in the presence of GM6001, a broad inhibitor of Matrix Metalloproteinases (MMPs) with an IC50 of 25.7 nM. This new, real-time, quantitative cellular analysis technique illustrates a novel method to simultaneously study migration and invasion of tumor cells. Citation Format: Meagan Roddy, John Rauch, Lindy O’Clair, Daniel M. Appledorn. Real-time, quantitative cellular analysis of migration and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5084.