The electrophoretic separation of myosin heavy chain isoforms from muscle biopsy homogenates has been widely practiced in the field of exercise physiology to examine how intrinsic (i.e., aging) and extrinsic (i.e., training) factors affect muscle phenotype. In the past, various research groups have used large and mini polyacrylamide gel systems to perform this delicate methodology. As technology has progressed, additional gel formats have been introduced, but available methodologies appear to be lacking. In this investigation, we successfully separated 3 distinct myosin heavy chain isoforms from various muscle samples using a modified mini gel system that can load up to 26 samples per gel. This article will outline our allocated protocol and discuss potential troubleshooting considerations for other researchers performing this intricate methodology. The outlined methodology has resulted in an ability to clearly resolute 3 distinct bands at molecular weights attributed to the myosin heavy chain isoforms in human skeletal muscle at a wide range of human ages (20-78 years). As additional technologies become available, the need to modify and adapt existing electrophoretic protocols for myosin heavy chain isoform separation and other protocols will continue to be evident.