Transfection is the introduction of foreign genetic material (e.g., DNA) into eukaryotic cells with the downstream goal of the expression and characterization of a protein with a specific biological function. This experimental technique is particularly useful in cancer research as it permits the study of oncogenes. When the foreign genetic material is incorporated into the genome of the host cell, then the technique is called “Stable Transfection.” Once the transfected DNA has been integrated, the gene of interest is permanently expressed, allowing for research to be conducted over extended periods of time without disruption. Here, we describe the lipid-based stable transfection of CD74 gene variants into the A549 lung adenocarcinoma cell line. CD74 is a type II cell surface receptor with a particular interest in cancer biology due to its key functional role in both solid and hematological malignancies. Our protocol provides a highly reproducible, step-by-step procedure for stable transfection that can be applied to a variety of genes.