Process development for transferring lab-scale research workflows to automated manufacturing procedures is critical chimeric antigen receptor (CAR)-T cell therapies. Thereby, the key factor for cell viability, expansion, modification, and functionality is the optimal combination of medium and T cell activator as well as their regulatory compliance for later manufacturing under Good Manufacturing Practice (GMP). In this study, we compared two protocols for CAR-mRNA-modified T cell generation using our current lab-scale process, analyzed all mentioned parameters, and evaluated the protocols’ potential for upscaling and process development of mRNA-based CAR-T cell therapies.