The majority of samples in forensic casework originate from sexual assault cases. The standard sexual assault examination kit includes multiple samples taken from various locations from the victim. In an effort to decrease the evidence processing time and reduce the number of untested sexual assault examination kits, many states, including Colorado, have passed new legislation to expedite evidence examination and DNA testing. Traditional examination methods of sexual assault evidence are time and labor intensive and often can only identify the presence or absence of biological material, such as semen and saliva. It is during this forensic biology examination that samples are taken for subsequent DNA analysis. The use of a male screen real-time quantification PCR assay has resulted in speeding up the process by eliminating the need to conduct forensic biology on a majority of sexual assaults. The male screen process can be used to determine if male DNA is present and if it is suitable for STR DNA analysis, possibly resulting in the identification of a suspect. The DNA present on these samples is typically a mixture of female and male contributors. Differential DNA extractions are utilized to attempt to separate the female and male DNA when spermatozoa (sperm) are present, but the traditional forms of differential DNA extraction are also time and labor intensive with results being sensitive to user experience. The optimized, semi-automated differential DNA extraction proposed in this study takes advantage of the Corning Life Sciences Costar® Spin-X® centrifugation filter tubes to prepare samples for extraction by speeding-up cell separation and lysis prior to extraction. This modification will result in samples conducive to automated extraction and purification on the Qiagen QIAsymphony SP robot. Prepared sample sets processed using the proposed modified extraction method will be compared with identical sample sets extracted with the current manual method in use at the Denver Crime Laboratory. The sensitivity, reproducibility and reliability of the modified system will be assessed, as well as the ability to extract varying levels of both female and male DNA in mixed samples. It is hypothesized that these modified methods will result in an improved DNA yield in a shorter amount of time. This optimized method for preparing samples for differential extraction is not specific for use with the Qiagen QIAsymphony SP robot, but is amenable for use with a variety of automated extraction robots and may be implemented into many different forensic laboratory systems.