The application of assisted reproduction 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 assisted reproduction techniques is currently based on criteria as viability, motility and morphology. The first sperm separation methods developed embraced washing procedures with subsequent resuspension of the male germ cells. Double density gradient centrifugation (DGC) and the swim-up procedure are currently used as standard preparation techniques while glass wool filtration (GWF) is also known to provide sperm samples with comparable recovery rates, motility, morphology, and fertilising capacity. Advanced protocols allow to select sperm according to their ultrastructural morphology or surface charges by electrophoresis. New insights into the molecular biology of spermatozoa prompted the development of molecular selection strategies. Those developments comprise hyaluronic acid mediated sperm selection, the 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 fertilise eggs using the hamster oocyte penetration assay.