Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products.