Mitochondria are the most important cellular structure for energy production, regulating ATP production through oxidative phosphorylation. Mitochondrial DNA (mtDNA) is translated by dedicated mitochondrial machinery to encode 13 polypeptides which are core subunits in creating ATP. Unfortunately, no safe and reproducible translation assay that can analyze defects in mitochondrial translation in depth has been fully developed. Thus, the objective of this work is to adapt pre-existing methodology to create a reproducible, semi-quantitative translation assay that can characterize the amount of production of each of the 13 polypeptides encoded by mtDNA. This assay can then be utilized to validate diagnosis, create new potentials for evaluating treatment of mitochondrial disease, and pave the way in creating the first clinically available assay for mitochondrial translation in the United States.