WERI-Rb1 retinoblastoma (Rb) cell line, a human photoreceptor model, is notoriously difficult to transfect. Culturing of the WERI-Rb1 cells as a monolayer is complicated and cells are easily detached during transfection. Furthermore, transfection efficiencies in monolayer and in suspension are moderate at best which has limited the analysis of photoreceptor-specific promoters with low activity. To overcome these limitations, we developed a straightforward reverse transfection method for WERI-Rb1 cells wherein snap-frozen DNA/polyethylenimine complexes are freeze-dried on the surface of 48-well plates and stored in desiccator until cells are seeded for transfection. Comparing to conventional transfection, reverse transfection turned out to have equal or better transfection efficiency. In addition, while conventional transfection with cationic polymers requires serum-free conditions, reverse transfection can be performed in the presence of serum. Importantly, DNA/polyethylenimine complexes promote cell adhesion to the plates. This enables cell culturing as monolayers with concurrent complex uptake. Also, long-term storage of the plates did not reduce the transfection efficiency nor it had any effects on the cell toxicity. Because of the stability of complexes, reverse transfection enables large-scale transfection of hard-to-transfect retinoblastoma cells thus providing a reproducible, cost-effective and versatile tool for parallel screening of proteins and gene regulatory elements used in diverse applications.