Spermatozoa have to be separated from seminal fluid to omit negative influences and to ensure best medically assisted reproduction (MAR) results. Several preparation techniques exist that can be used to enrich the best motile and morphologically normal spermatozoa: simple washing, swim-up (with or without washing step) and density gradient centrifugation. Every technique has its advantages and disadvantages, and also, the final attribution of the sample (e.g., usage for intrauterine insemination, in vitro fertilization or intracytoplasmic sperm injection) has to be taken into account. Simple washing is optimal for high sperm numbers in normozoospermic patients. Swim-up gives excellent results in normozoospermic and moderate oligoasthenoteratzoospermic (OAT) samples, and density gradient centrifugation is preferred if a sample is moderate to severe OAT. Epididymal aspirated samples can be prepared either by swim-up or by density gradient. Testicular sperm extraction samples have to be processed differently, as steps dissociating the tissue have to be executed first. Good-quality sperm preparation is a prerequisite for the highest success rates in MAR.