Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short‐tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non‐sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post‐coitus (PC), the new method identifies single‐source (male) STR profiles. When applied to samples collected 24–72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre‐processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.