This chapter discusses the techniques, methodology, and procedures that are highly relevant to the current practice of assisted human reproduction and sperm banking. Cryopreservation of sperm cut down the necessity of obtaining fresh sperm for subsequent assisted reproductive technology (ART) cycles. Abundant evidence exists in literature indicating that frozen sperm are as good as fresh sperm in fertilizing oocytes and subsequent developments. The success of cryopreservation is affected by many factors including membrane permeability, amount of intracellular water, type of cryoprotectant, and the method of freezing and thawing. Four cryoprotectants that are most often used for cryopreservation of human spermatozoa are: glycerol, dimethylsulfoxide (DMSO), ethylene glycol, propanediol, egg yolk and buffering agents. The American Society for Reproductive Medicine (ASRM) practice committee recommends evaluations of potential sperm donors incorporating recent information about optimal screening and testing for sexually transmitted infections, genetic diseases, and psychological assessments.