Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are promising candidates for cancer immunotherapy, but their development is impeded by time-consuming and costly virus-based methods for CAR validation. Plasmid electroporation offers a potentially faster and more cost-effective alternative; however, its use is limited by severe toxicity to NK cells, resulting in poor viability and low transgene expression. To overcome this limitation, we performed genome-wide CRISPR knockout screening and uncovered the sensing pathway (details not disclosed due to pending patent applications) as the key mediator of plasmid toxicity in NK cells. By using pharmacological inhibitors and CRISPR gene editing, we confirmed that blocking this pathway alleviates toxicity, enabling robust transgene expression from plasmid. Building on this discovery, we developed a plasmid-based high-throughput CAR screening platform using a sensor-deficient NK-92 cell line, allowing for rapid and efficient CAR optimization. By integrating this platform with phage display technology, we screened scFvs targeting EGFR and EpCAM and identified several novel CAR constructs. The top-performing EpCAM CAR constructs were further validated through lentiviral transduction in primary NK cells, demonstrating potent anti-tumor activity in in vitro assays and in vivo xenograft model. These results confirmed that CAR constructs identified via our plasmid-based platform retain efficacy when transitioned to viral systems. By overcoming the challenge of plasmid-induced toxicity, our rapid CAR-NK platform bridges early-stage research with preclinical development, complementing traditional virus-based methods. This innovative approach accelerates CAR-NK optimization by reducing costs and timelines to develop more potent NK cell therapeutics. Our work overcomes key challenges in CAR-NK development, paving the way for more efficient and scalable cancer immunotherapies.