The application of assisted reproductive techniques (ART) has provided help to many men seeking to father a child, although the current success rates of these procedures remain suboptimal. Selection of human spermatozoa prior to ART currently is based on criteria that include viability, motility and morphology. The first sperm separation methods to be developed involved washing procedures with subsequent resuspension of the male germ cells. Double-density gradient centrifugation (DGC) and the swim-up procedure are currently standard preparation techniques. Glass wool filtration (GWF) is also known to provide sperm samples with comparable recovery rates, motility, morphology, and fertilizing capacity. Advanced protocols allow for selecting sperm according to their ultrastructural morphology or surface charges by electrophoresis. New insights into the molecular biology of spermatozoa have prompted the development of molecular selection strategies, including hyaluronic acid-mediated sperm selection, annexin V magnetic- activated cell separation (MACS), and annexin V molecular glass wool filtration. Sperm preparation that combines MACS with double-density centrifugation provides spermatozoa of higher quality in terms of motility, viability and apoptosis indices compared with other conventional sperm preparation methods. Furthermore, sperm prepared according to this protocol showed improved ability to fertilize eggs using the hamster oocyte penetration assay.