This chapter discusses methods of preparation of spermatozoa for intrauterine insemination (IUI) as well as other assisted reproductive technologies (ART) techniques. Freshly ejaculated sperm are incapable of fertilization and must undergo a series of changes in structure and chemical function, termed capacitation, which end in the ability of the sperm to successfully interact with and fertilize the oocyte. Transport of spermatozoa from the male to the site of fertilization in the female involves both active and passive transport processes from the seminiferous tubules through the epididymis and vas deferens, ejaculation into the vagina, and contact with the cervix in the female. In semen processing, the physical characteristics of some semen samples may interfere with the efficiency of the separation procedure, e.g. seminal viscosity, incomplete liquefaction, and excessive numbers of cells other than spermatozoa. The simple wash procedure is recommended for semen specimens with low sperm count and motility.