Background: The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction.Objective: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets.Methods: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells.Results: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts.Conclusion: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.