Significance An emerging area of synthetic biology is the engineering of bacteria to diagnose and treat various diseases in the body. However, the lack of physiologically relevant in vitro testing environments to rapidly screen bacterial therapies limits their development for clinical use. Here, we develop a platform that enables parallel and long-term monitoring of engineered bacteria in multicellular spheroids. Using this system, we rapidly screened tumor-targeting bacteria engineered to deliver a library of anticancer molecules via synthetic gene circuits. We demonstrate high similarity between in vitro and in vivo results and show broad applicability of the system with various bacterial species and cell types. This technology may serve to accelerate future clinical applications for synthetic biology.