The need to increase throughput in forensic DNA genotyping has led to a growing interest in developing new DNA quantification methods that are more efficient than the slot blot hybridization method currently used in many forensic DNA labs. One such method that is proving useful for forensic DNA qualifications is real-time quantitative PCR (qPCR), a method widely used in biomedical research and molecular diagnostics. This report describes a duplex real-time qPCR assay for the simultaneous quantification of human nuclear and mitochondrial DNA in forensic samples. The assay was designed to be of general utility for forensic DNA quantifications and specifically useful for the post-extraction analysis of samples that contain highly degraded DNA. A new nuclear-mitochondrial duplex qPCR assay is described, however alternative nuclear and mitochondrial target sequences for amplification and quantification were chosen. A target sequence was chosen that spans the repeat region of the primate-specific TH01 STR locus which has been widely used for forensic applications. The report provides a sensitive means for determining the presence of human mitochondrial DNA in forensic samples. For quantifying DNA in highly degraded samples, the results indicate that the length of the qPCR target sequence is an important consideration when selecting an assay for forensic use. Figures, tables, and references