The elimination of infected or cancerous cells by CD8+ cytotoxic T lymphocytes (CTL) is a crucial effector mechanism of the immune system. Upon antigen recognition, CTL stop migrating, establish a tight contact with target cells and deliver cytotoxic molecules such as perforin and granzymes that lead to target cell apoptosis. The ability of CTL to control a population of infected cells or a tumor depends on multiple parameters, such as the relative numbers of CTL and target cells, the intrinsic cytotoxic activity of CTL, the intrinsic resistance of target cells and the repertoire of immune checkpoints tuning cytotoxic activity at the CTL:target cell interface. In this context, in vitro assays to precisely measure CTL:target cell interactions and cytotoxic activity over time are required to monitor natural or therapeutic responses. We here present an image-based method that allows recording of positions and survival of CTL and target cells over time in a high-throughput format. The protocol relies on the staining of CTL and target cells with fluorescent dyes and the automated imaging of cells deposited in wells of a 384-well plate with an automated cell imaging device. We discuss potential applications offered by the kinetic assessment of CTL cytotoxic activity in a high-throughput format.