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Background Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as an advanced and highly effective approach for treating hematologic malignancies with its scope rapidly expanding to encompass diverse applications including solid tumors. This fast-paced development calls for highly sensitive and quantitative ex vivo assays for assessing CAR-T cell cytotoxicity, particularly in models that recapitulate 3-D tumor environments.Methods To address this need, we generated GFP-luciferase dual reporter cancer cell lines that endogenously express high levels of key CAR-T target antigens, CD19 and BCMA. Using both antigen-specific and mock-engineered CAR-T cells, we assessed cytotoxicity via multi-modal imaging strategy, integrating luciferase-based bioluminescence assay with phase contrast and fluorescence live cell imaging. To further demonstrate dynamic visualization of CAR-T activity in an environment that mimics in vivo conditions, we embedded dual reporter spheroids in 3-D matrices which allowed for time-lapse imaging of CAR-T cell infiltration.Results Our multimodal imaging approach revealed significantly enhanced cancer cell killing by targeted CAR-T cells compared to controls. Employing 2-D and 3-D co-culture assays of dual reporter cancer cells with targeted and mock-engineered CAR-T cells, we found that targeted CAR-T cells exhibited higher killing of cancer cells than mock CAR-T cells in both live imaging and luciferase assays. In 3-D fluorescence imaging experiments, we identified a reduction in both the size of cancer cell spheroids and in the GFP signal in response to targeted CAR-T cells as compared to mock CAR-T cells. Additionally, in both 2-D and 3-D co-culture assays, we noticed a significant decrease in relative luciferase signal from reporter cancer cells in conditions with targeted CAR-T cells, implying targeted cancer cell killing by CAR-T cells. Moreover, time-lapse confocal imaging of spheroids in 3-D matrices captured active infiltration of CAR-T cells into tumor spheroids over time.Conclusions These results showcase the benefit of multimodal methodology combining bioluminescent and live fluorescence imaging to quantitatively examine CAR-T cytotoxicity as well as visualize the spatial and temporal interplay of CAR-T cells and cancer cells in both 2-D and 3-D co-culture systems. The scalability and sensitivity of this assay platform makes it a versatile tool for standardizing CAR-T cytotoxicity evaluation, advancing CAR-T therapeutic development in industrial and translational research settings.