The ability to observe and quantitate T cell-mediated tumor cell killing at the individual cell level is critical for understanding the mechanism of immune activation and exhaustion to assist therapeutic designs. In order to acquire large amount of single cell data for statistical analysis, images of cells in micro-gridded chambers (sub-wells) within a standard subwell are captured to analyze single cell interactions. The current available gridded platforms, manual microscopy imaging or standard automated image cytometer methods however are time-consuming. In addition, the analysis software is typically used for custom-made sub-wells constructed with Polydimethylsiloxane. In this work, we reported a high-throughput single T cell killing assay utilizing the Celigo Image Cytometer and Elplasia SQ plates that convert each 384-well into 86,400 sub-wells. In this assay, both T cells and tumor cells were seeded with varying densities, resulting with 1 to 100 cells in each sub-well, as well as varying T cell activation reagent. The plate was scanned immediately after cell seeding at t = 0, 4, 22, 46, 68, and 168 hours, where the number of cells were tracked in each individual sub-well over time for each cell type. The image cytometer was able to rapidly acquire and analyze images at 1 µm2/pixel. The images and results were exported into a custom program to determine the proper sub-well location of the segmented cells to allow separate tracking of small groups of tumor and T cells. This approach gave the image cytometry method greater capability in resolving subpopulations within the biological sample, resulting in more detail on the cytotoxic killing as a function of cell demographics in the tumor microenvironment.