Coenzyme Q 10 (CoQ 10) deficiency syndrome includes clinically heterogeneous mitochondrial diseases that show a variety of severe and debilitating symptoms. A multiprotein complex encoded by nuclear genes carries out CoQ 10 biosynthesis. Mutations in any of these genes are responsible for the primary CoQ 10 deficiency, but there are also different conditions that induce secondary CoQ 10 deficiency including mitochondrial DNA (mtDNA) depletion and mutations in genes involved in the fatty acid β-oxidation pathway. The diagnosis of CoQ 10 deficiencies is determined by the decrease of its content in skeletal muscle and/or dermal skin fibroblasts. Dietary CoQ 10 supplementation is the only available treatment for these deficiencies that require a rapid and distinct diagnosis. Here we review methods for determining CoQ 10 content by HPLC separation and identification using alternative approaches including electrochemical detection and mass spectrometry. Also, we review procedures to determine the CoQ 10 biosynthesis rate using labeled precursors.