During the anti-tumour immune response, cytotoxic T lymphocytes (CTLs) can target and induce apoptosis in cancer cells. However, the killing function of CTLs can be perturbed directly by cancer cells or the tumour microenvironment (TME). Among the various factors in TME, the effect that mechanical properties of the extracellular matrix (ECM) have on CTL responses is unclear. Research into CTL-mediated killing is typically performed in either two-dimensional (2D) matrix-free culture or in complex in vivo animal models. In vitro, 2D studies are limited in recapitulating the CTL response in vivo, whereas it is very difficult to manipulate the TME and perform high-throughput experiments using in vivo models. Recently 3D culture models have been introduced to fill the gap between 2D and in vivo studies. In this study, we used an automated 3D bioprinter to incorporate OT-I T cells and cognate and non-cognate target cells in a polyethylene glycol (PEG)-based hydrogel and studied the killing efficiency in comparison with 2D culture and manually-prepared gels. Without compromising cell viability, 3D bioprinter embeds both CTLs and target cells in the hydrogel and enables control over the dimensions of the embedding matrix as well as the number and spatial organisation of cells. Moreover, the ability to digest the gel and release the cells allowed us to perform killing efficiency comparisons and downstream high-throughput CTL functional analyses using flow cytometry. In parallel, to compare CTL response in 2D and 3D cultures, we visualised the killing potential through high-throughput confocal imaging. Finally, this novel 3D cell culture system allowed us to investigate the effects of tunable ECM mechanical properties in a reproducible killing model of matrix embedded CTL and target cells.