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Summary: This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of human CTL behavior, from adhesion to diapedesis, using live cell imaging which cannot be achieved by in vivo imaging. The flow system is made of 2D plastic surfaces covered by an endothelial monolayer limiting the system but allows for quantitative analysis of CTL behavior with high modifiability.For complete details on the use and execution of this protocol, please refer to Schoppmeyer et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.