The test developed uses the cytochrome b (cytb) and the ribosomal 12S genes on the mitochondrial genome. Universal and human specific fragments of similar size are amplified and quantified using SYBR Green. The human fraction of a sample can be subtracted from the universal fraction for an accurate estimation of nonhuman mtDNA copy number. The test was validated with 24 human samples and 27 nonhuman mammalian samples. The test is applicable to ancient or highly degraded samples. Human contamination in animal samples can be determined and diluted to a level that will not interfere with sequencing reactions. The method is fast, simple, and cost-effective while permitting the accurate quantification of mtDNA copies from a wide range of mammalian species regardless of the tissue type. The method can be adapted for use in detection, separation, and quantification of other biological samples with the design of specific primer sets. The descriptions of materials and methods addresses primer design, standards, and sample testing. 5 tables, 4 figures, and 37 references