The project successfully developed a robust qPCR assay for the simultaneous detection and quantification of total human genomic and male DNA for use in forensic DNA analysis. The precision, accuracy, specificity, and low limits of detection of the assay were demonstrated. This process is a superior alternative to the slot blot methods commonly used in forensic laboratories as well as other commercially available qPCR assays. The accuracy of the assay was better than the slot blot method and similar to the Quantifiler kits. The duplex qPCR assay provides a significant time-savings compared with the slot blot methods. Whereas the slot blot method requires 3 or more hours of hands-on time, the qPCR requires less than 1 hour of manual setup, followed by an automated reaction of approximately 2 hours. In addition, DNA quantitation from the qPCR is automatic; slot blots require visual examination or transfer to an imager for DNA quantitation. The simultaneous quantitation of male and total human genomic DNA gives the user several advantages over singleplex reactions. The description of materials and methods encompasses primer/probes, DNA samples, real-time PCR assay, the slot blot procedure, the quantifiler comparison, and the mixture challenge. 2 tables, 5 figures, and 19 references