An optimized protocol for the electroporation-based transfection of tobacco protoplasts is described that routinely results in transgene expression frequencies approaching 90%. The overall efficiency of the procedure depends collectively on numerous key parameters, including protoplast viability; DNA concentration, purity, and topology; carrier DNA; and electrical conditions such as ionic strength of the electroporation buffer, electric field strength, pulse duration, and capacitance. Individual methodologies that address each one of these parameters are presented in sufficient detail to enable successful reproduction of this method along with notes that describe helpful tips.