The prediction of sperm fertility is of paramount importance for breeding animals. Multiple laboratory approaches have been developed for this purpose, but they have yielded equivocal results. The objective of this study was to develop and standardize to a method for predicting fertility in vivo in boars using the in vitro penetration assay. To increase the sensitivity and reduce false-negative results of the assay, each step in the procedure was standardized and quality control was applied. Maximum penetration of hamster zona-free oocytes and immature porcine oocytes was obtained using heparin-treated sperm cells. Hamster zona-free oocytes showed a significantly higher penetration than immature porcine oocytes. To eliminate interassay variability, two frozen bull semen samples were applied. All possible variables related to the female were excluded. The SPA (sperm penetration assay using zona-free oocytes) result showed significant correlation with historic average litter size but had no significant correlation with farrowing rates. To determine the normal range for the SPA, lower limits of the sperm fertility index were established as 1.2 for the small litter sizes (<8 piglets) and 2.5 for the large litter sizes. The overall accuracy was 92 and 96% respectively, for the small and large litter sizes. Our laboratory has standardized the procedure for the SPA, resulting in greatly increased sensitivities for small and large litter sizes. The protocol increases the ability to discriminate between good and poor fertility groups and it was highly effective at ranking 24 boars by litter size into large and small litter groups.