AbstractPolyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI‐based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI‐based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI‐based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc.