Introduction: With better appreciation of the tumour microenvironment and success of immune checkpoint inhibitors (ICIs) in locally advanced rectal cancer (LARC), there is growing interest to explore novel immune-based therapies. Chimeric antigen receptor (CAR) T cell therapy is a form of adoptive cell therapy which is highly effective in haematological malignancies but results are disappointing in solid tumours. We aimed to explore its potential in LARC by developing a preclinical assay using a rectal cancer organoid CAR-T cell co-culture model. Methods: Rectal cancer biopsies were obtained from two patients at Peter MacCallum Cancer Centre, from which organoid cultures were established. Lewis Y (LeY), a difucosylated carbohydrate antigen which is overexpressed in colorectal cancer, was selected as the CAR target and confirmed to be positive in these organoids. Co-culture assay was performed over 48 hours. Cytotoxicity of LeY CAR-T cells was measured using a fluorescence-based imaging method. ICIs, in the form of PD-1 inhibitors, were added to assess for synergistic effects. Results: In both patients, our assay detected dose-dependent cytotoxicity of LeY CAR-T cells against its target antigen, with significant reduction in fluorescence signal intensity, and monitored killing response over time. The addition of PD-1 inhibitors did not affect CAR-T cytotoxicity. In addition, we were able to provide mechanistic insight into the CAR-T cell response through RNA sequencing and flow cytometry. Conclusion: Our assay provides a robust and high-throughput method to test and optimise CAR-T cell therapy before in vivo testing in LARC. It can become a useful tool for both basic CAR-T cell therapy research and personalised medicine.