An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613‐bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co‐amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real‐time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 ± 1.01 × 10⁴ molecules/ng total genomic DNA using competitive PCR vs 4.90 ± 0.84 × 10⁴ molecules/ng total genomic DNA using real‐time PCR), both inter‐ and intraexperimental variance were significantly lower using the real‐time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real‐time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage‐ and depletion‐associated disorders. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:180–186, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20024