Transfection of therapeutic genes to retinal pigment epithelial cells has many potential applications for the treatment of ocular diseases. This protocol has been systematically optimized to obtain reproducible, high-level gene transfer to retinal pigment epithelial cells (ARPE-19) in vitro, using branched polyethyleneimine (PEI) as a gene carrier and secreted Renilla Luciferase as a reporter protein. In a first stage of the protocol development, different parameters, including cell density at seeding, PEI/DNA charge ratio, composition of the preparation buffer and nanoparticulate assembly conditions as well as incubation time of the nanoparticulates with ARPE-19 cells, were optimized in a matrix-like fashion. Selection of the most effective conditions for gene transfer led to the finalization of the present protocol, with which transgene expression efficacies of 10-20 ng/ml are typically obtained. Cell viability is 60-80% depending on the incubation time of the nanoparticulates with cells. This protocol has been optimized for ARPE-19 cells and for PEI as gene carrier. However, with minor changes, it should be suitable also for transfection of other cultured cells, as well as for different polymeric carriers.