In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving case works where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red-Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological above-mentioned variables.