Gold nanoparticles (AuNPs) coated with PEI-based copolymers have emerged as a promising non-viral vector for gene therapy due to their exceptional biocompatibility and efficient gene delivery capabilities. Green synthesis methods, which utilize environmentally friendly and sustainable approaches, have gained significant attention for producing biocompatible nanoparticles. While AuNPs have been extensively studied, challenges often arise when conducting cell viability assays, particularly using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The inherent properties of nanoparticles, such as their size, shape, and surface chemistry, can interfere with the MTT assay's mechanism, leading to inaccurate assessments of cell viability. Additionally, the interaction between the MTT reagent and the nanoparticles can further complicate the assay and potentially yield misleading results. To address these challenges, this study aims to optimize AuNP synthesis, establish optimal MTT assay conditions and mitigate interference effects by carefully controlling the synthesis parameters, optimizing the MTT assay conditions, and addressing the potential interference effects. This study aims to provide a reliable and accurate method for evaluating the cytotoxicity and biocompatibility of AuNP-based gene delivery systems. This knowledge will contribute to the development of safe and effective nanomedicine for various biomedical applications.