Abstract Differential lysis is an established method for vaginal swabs containing semen stains. However, it can be a challenge to obtain a single-source male profile when vaginal swabs contain only low amounts of spermatozoa, as DNA from the perpetrator's sperm cells tends to be obscured by the presence of an excess of DNA from epithelial cells. In our study we compared seven different protocols (Differex™, Sampletype i-sep ® DL, Sampletype i-sep ® SQ, GEN-IAL ® First-DNA all tissue kit, The Erase Sperm Isolation Kit, QIAcube Washing Station, in-House-method) in order to determine the efficiency of sperm separation and DNA yield. In a first evaluation, a high concentration of semen (about 1000ng) was transferred onto vaginal swabs. The changes of the female:male ratio in the sperm/non-sperm fractions comparing the standard extraction samples were used to determine efficiency of separation. The recovery of spermatozoa was determined based on male DNA concentration in the samples. Some methods showed significant improvement in the female:male ratio as well as the high male DNA recovery and were therefore subsequentially investigated using decreasing amounts of semen.