DNA isolated from crime scene samples must be quantitated to determine the amount of human DNA present. In attempting to develop faster, cheaper, and more quantitative methods, the forensic community has identified real-time PCR methods as having the advantages of being fast and quantitative with much less hands-on time than prior slot blot methods. This study describes the development and optimization of a MGB Eclipse assay, the Alu sequence-based assay, a real-time PCR method for the quantitation of human DNA. Most experiments were performed with a human DNA standard purchased from Promega in Wisconsin. DNA was isolated from a number of samples to evaluate the assay: blood spots from five DNA databank samples, five samples from control blood spotted on denim or colored cloth, control blood placed on five surfaces, a control blood spot placed in the dark for 3 months, two male and two female fractions from sexual assault cases, and two samples from a proficiency test. The primary advantage found for the MGB Eclipse real-time Alu assay was its much greater dynamic range. The Eclipse assay has the advantage of lower cost than the commercial kits and the greatest sample concentration range of published assays, additionally; it has simplicity, speed, less hands-on-time, and automated quantitation. Figures, tables, and references