Mitochondria are the most important cellular structure for energy production, generating ATP production through oxidative phosphorylation. Mitochondrial DNA is translated by dedicated mitochondrial machinery to encode 13 polypeptides which are core subunits in creating ATP. Unfortunately, no reproducible and fully quantitative translation assay that can analyze defects in mitochondrial translation has been developed. Thus, the objective of this work is to utilize stable isotope labeling by amino acids in cell culture (SILAC) to create a reproducible and quantitative mitochondrial translation assay that can characterize the amount of production of each of the 13 polypeptides encoded by mtDNA and be potentially translatable as a clinical assay. 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.