Autologous chimeric antigen receptor (CAR)-T therapies have shown exceptional efficacy against hematological malignancies and potential against various diseases. However, robust in vitro efficacy and safety tests (ESTs), crucial for their clinical application, are lacking. Here, we developed α-PD-L1- natural killer (NK) cells by engineering NK92 cells to express a CAR targeting PD-L1, and selected three lead CAR-NK cell lines. Using an in vitro EST that integrates direct/indirect cytotoxicity and soft agar formation assays, we identified a candidate CAR-NK cell line (α-PD-L1-#2L5-NK). To validate the EST, we assessed the in vivo efficacy of α-PD-L1-#2L5-NK in a murine xenograft model, demonstrating significant tumor size and weight reduction. Additionally, α-PD-L1-#2L5-NK did not induce mortality or toxic effects in mice. Overall, this EST is valuable for screening and identifying safe and effective cell therapies in the off-the-shelf immune cell therapy field. It can help reduce the likelihood of failures of therapeutics and establish standards for quality control and regulatory aspects.