Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called "Design of Transfections" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.