The primary objective of this study was to establish whether a combination of pre-insemination stallion sperm parameters can be used to accurately predict pregnancy outcome. This study was based on 46 inseminations of 41 mares, by a cohort of 7 standardbred stallions over a 5-week period at an Australian pacing stud. For the analyses, semen quality was assessed immediately after collection, and again after the semen had been stored at 5 oC for 24 h in EquiPlus semen extender. Following semen collection and chilling, assessments of ejaculate volume, sperm concentration, and motility were performed using an iSperm® Equine portable device. Additionally, an aliquot of the semen sample was subjected to a migration assay through a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15 min period. This subpopulation of isolated cells were again assessed for concentration and motility. Furthermore, the ability of the isolated spermatozoa to reduce the membrane impermeant tetrazolium salt WST-1 was evaluated. This information, combined with data that described the stallion and mare ages were used as predictors of pregnancy outcome, as confirmed by rectal ultrasound performed 14 days post ovulation. The predictive criteria used to predict pregnancy via multivariate discriminate analyses were optimized for each individual stallion, resulting in the ability to predict pregnancy outcome following insemination with an overall accuracy of 87.9% if analysed pre-chilling, and 95% if analysed post-chilling. The application of such an approach to semen analysis would be of considerable value to the equine ART industry in managing the reproductive performance of its stallions.