The study determined that the Quantifiler Duo DNA Quantification Kit enabled quantification of total human DNA and human male DNA as well as detection of the presence of PCR inhibitors in biological samples in a single amplification reaction. In addition to the quantification of DNA, the assay provides a quality assessment of forensic evidence samples in terms of determining male to female DNA mixture ratios and estimating the extent of PCR inhibition. The information is useful in selecting the appropriate AmpFl STR amplification chemistry for obtaining an interpretable short tandem repeat profile in the first attempt. The developed assay consumes a minimal quantity of the forensic sample. The developmental validation studies were performed following the revised validation guidelines provided by the Scientific Working Group on DNA Analysis Methods. Pooled human male genomic DNA used for the generation of standard curves was obtained from EMD Biosciences, Inc. Genomic DNA from unknown individuals was obtained from a variety of commercial sources. The descriptions of materials and methods addresses the extraction and quantitation of DNA real-time PCR amplification, STR analysis, sensitivity study, species specificity, precision and accuracy, reproducibility, mixture study, calculation of male to female DNA ratio, inhibited sample, degraded DNA, and case-type samples. 9 tables, 11 figures, and 15 references