The cryopreservation of bull semen adversely affects the metabolism, motility and membrane integrity of the spermatozoa, thereby decreasing the fertilizing ability of the processed sample. The present review covers methods available for examining the functionality of bull spermatozoa after thawing, including the use of fluorophores in combination with morphological examination. Procedures for clean-up, separation and selection of morphologically-normal, viable spermatozoa in vitro are also reviewed. Among the reviewed procedures are methods for dilution and washing and the selective fractionation of sperm subpopulations (especially density-gradient centrifugation in silica-bead suspensions), as well as methods based on adherence to glass (differential filtration) and sperm self-migration (particularly swim-up through hyaluronic acid). Emphasis is placed on assessing the value of these techniques for diagnostic purposes and for optimizing the methodology ofin vitro fertilization.