Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 mu g) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.