Forensic DNA analysis of sexual assault evidence requires separation of DNA from epithelial (victim) and sperm (perpetrator) cells. The conventional method used by crime laboratories, which is termed "differential extraction", is a time-consuming process. To supplant the conventional process, separation of sperm from a biological mixture containing epithelial cells has been demonstrated on a microfluidic device. This separation utilizes the differential physical properties of the cells that result in settling of the epithelial cells to the bottom of the inlet reservoir and subsequent adherence to the glass substrate. As a result, low flow rates can be used to separate the sperm cells from the epithelial cell-containing biological mixture. Following cell separation on the microdevice, DNA extraction, amplification, and separation were performed using conventional laboratory methods, showing that the cell separation product in the outlet reservoir was of male origin. The reported cell separation has the potential to impact the forensic DNA analysis backlog of sexual assault cases by circumventing the time-consuming conventional differential extraction procedure.