The inner perivitelline layer (IPL) sperm hole assay provides an objective assessment of the number of sperm penetrating the hen's ovum at the germinal disc. That number will provide insight into numerous parameters concerning fertility, including, but not limited to, the probability of the ovum being fertilized (true fertility), the duration of fertility, an assessment of mating activity, the effect of inseminating in vitro-stored semen, and the influence of spiking a breeder flock. In the current paper, we discuss the use of a filter paper ring to isolate and process the IPL overlying the germinal disc (PL disc) in 5 species of domestic birds. The morphology of the PL disc is also described to assist those using the sperm hole assay during sperm hole counting. The filter ring approach was successfully used with chicken, turkey, duck, goose, and Japanese quail eggs; the PL discs remained intact and stretched across the filter ring and sperm holes, when present, were clearly discernible. Morphologically, the PL disc appeared to be a silhouette of the germinal disc, including the germinal vesicle observed in follicular oocytes. The fibers forming the IPL had a honeycomb-like arrangement that could be mistaken for actual sperm holes in faintly stained slides. Given its diversity of applications and relative simplicity using the filter ring, it is recommended that the sperm hole assay be part of a quality control program at the hatchery. Already used in the scientific community, this filter ring method has the additional advantage that the blastoderm may be collected before the staining process. © 2014 Poultry Science Association, Inc.