Typing of sperm DNA found in vaginal fluid is an important component of investigations of sexual crimes. A standard, two-step differential extraction of sperm and vaginal epithelial cells from vaginal fluid mixed with semen is usually used for DNA isolation. However, this method is considerably time-consuming and, due to often insufficient separation of sperm and vaginal pithelial DNA in the centrifugation process, DNA contamination may occur. Therefore, we have developed a new and more reliable method of sperm DNA extraction from vaginal fluid. Using a three-step, complete digesion of vaginal fluid with deoxyribonuclease I (DNase I) we were able to isolate pure sperm DNA in a shorter time. It can be expected that this improved method of sperm DNA isolation will lead to more reliable DNA typing and, significantly improve the efficiency of scientific criminal investigation such as DNA typing.