The detection of semen can assist in reconstructing the events of a sexual assault and impact the outcome of legal dispositions. Many methods currently are used for detecting the presence of semen, but they all have limitations with regards to specificity, sample degradation/consumption, stability of biomolecule assayed, and/or incompatibility with downstream individual identification assays. DNA is routinely collected at sexual assault crime scenes and is widely used for individual identification. The DNA also carries methylation patterns that are tissue specific. To date, however, assays designed to exploit methylation patterns suffer from complex chemistries and unwieldy analyses. DSI-Semen™ kit uses a novel approach involving CpG methylation-sensitive restriction endonuclease digestion coupled to a multiplexed polymerase chain reaction (PCR) to generate an amplicon profile that makes it possible to determine whether the tissue source of a DNA sample was semen or non-semen. The assay returned an appropriate positive result for semen with neat semen, semen stains, and semen/non-semen tissue mixtures. The assay is robust and reliable, with a positive result for semen given as little as 31 pg of template DNA input. Low levels of semen were detected in mixtures of semen and other body fluids. UV-exposed samples and those in the presence of limited concentrations of known PCR inhibitors were typeable. The DSI-Semen™ kit provides a reliable tool for the determination of DNA being derived from semen.