Research on chimeric antigen receptors (CARs) has advanced rapidly, demonstrating efficacy in cancer treatment. However, there still exists an important limitation in the development of cell therapy products, as considering time is required to develop and validate target single-chain variable fragments (scFvs) against antigens in various cancer types. To rapidly screen and validate more effective antigen-specific single-chain variable fragments (scFvs), we utilized mRNA-based CAR-NK cells to evaluate the therapeutic efficacy against mesothelin (MSLN)-expressing cancer cells. MSLN is a cell surface protein highly expressed in many cancer types, but its distal portion can be cleaved by proteases, resulting in high concentrations of soluble MSLN (sMSLN) in the tumor microenvironment. This has been reported as a key obstacle for antibody-based therapies and anti-MSLN CAR therapies. We identified scFv candidates that could effectively target the truncated form of MSLN and evade the shedding effect of sMSLN, demonstrating potent anti-tumor activity. This approach enables the rapid screening and selection of scFv candidates based on their specific binding characteristics and efficacy against cancer-specific antigens, which can significantly accelerate the development of effective antigen-targeting CAR therapies.