Precise assessment of the cytotoxic activity of engineered immune cell therapeutics, such as chimeric antigen receptor-engineered T (CAR-T) cells, is essential for their development and quality control. However, luciferase (Luc)-based viability assays, which evaluate target cell viability by overexpressing Luc tags and measuring chemiluminescent signals, may yield biased results depending on the promoter driving Luc expression. This study demonstrates that CAR-T cells can enhance cytomegalovirus (CMV) promoter-driven transcription in target cells via the interferon-gamma (IFN-γ)/nuclear factor κB (NF-κB) signaling pathway, leading to elevated Luc expression and a discrepancy between chemiluminescent signals and actual target cell death. These findings underscore the limitations of CMV promoters in functional protein overexpression systems in the context of engineered T cell killing of target cells due to their susceptibility to transcriptional interference. Statistical analyses indicate that Luc expression driven by the elongation factor-1 alpha (EF1α) promoter exhibits the highest concordance with flow cytometry-based quantification across three CAR-T cytotoxicity assay platforms, making it a more reliable choice for evaluating CAR-T cell cytotoxicity. This study highlights the necessity of selecting appropriate promoters to ensure accurate Luc-based cytotoxicity assessments and provides critical insights for standardizing detection methodologies in CAR-T cell evaluation.