The overexpression of a specific protein is a common method for investigating the specific biological function of the substance and the mechanism of action. In vivo electrotransfer has been confirmed to be one of the most reliable, efficient and cost-effective way to overexpress a protein in a select biological tissue. Typically, this technique involves a physical injection of plasmid DNA followed by electric pulses across the injection site. Here, we introduce this method that we used to transfect green fluorescent protein (GFP)-tagged PGC-1α plasmid DNA into mouse tibialis anterior (TA) muscle, which attained high transfection efficiency with no muscle damage. To quantify the transfection efficiency, we also demonstrate the visualization of plasmid DNA transfected fibers via immunohistochemical staining on muscle cross sections.