Muscle tissues attached to the proximal ends of hamstring tendon grafts are routinely discarded during anterior cruciate ligament (ACL) reconstruction. Preserving the muscle on the ACL graft was shown to improve the surgery outcomes, possibly due to the osteogenic differentiation of muscle satellite cells, accelerating graft integration into the underlying bone. The enhancement of muscle cell osteogenic differentiation was previously demonstrated via adenoviral transfer of bone morphogenetic protein-2 (BMP-2). We evaluated gene electrotransfer as a fast, non-viral and clinically relevant alternative for the delivery of plasmid DNA to muscle-tendon ACL grafts. Human muscle-tendon tissues remaining were collected from 13 patients undergoing ACL reconstruction procedures, and standardized tissue samples were injected twice with 20 µg plasmid DNA or not treated. A combination of high voltage (600 V, 100 µs) and low voltage (80 V, 100 ms) electric pulses or medium voltage (MV; 200 V, 20 ms) pulses were first tested using plasmid DNA encoding the green fluorescent protein. The selection of MV protocol was confirmed with a luciferase plasmid and subsequently used to test a therapeutic BMP-2 plasmid. Upon detailed evaluation of individual tissue sample properties (i.e., donors, thickness and volume) and their BMP-2 release, further optimization of tissue selection and preparation for gene electrotransfer was defined. This study indicates the feasibility of gene electrotransfer as a method to deliver plasmid DNA easily and rapidly to muscle tissue preserved on ACL grafts.