Banked serum samples from seven okapi (Okapia johnstoni) with known pregnancy status were evaluated using the BioPRYN wild enzyme-linked immunosorbent assay to detect pregnancy-specific protein B (PSPB). Thirty-six serum samples, 18 from known pregnant and 18 from nonpregnant okapi, were analyzed. Using optical density cutoffs, the BioPRYN wild assay demonstrated a sensitivity of 88% (95% confidence interval, 65%–98%) and a specificity of 100% (95% confidence interval, 81%–100%). In one sample, this test confirmed pregnancy as early as 21 days of gestation; however, two pregnant okapi were reported to be not pregnant at 23 and 38 days of gestation, suggesting sensitivity may be lower in early gestation. Sensitivity improved to 100% when samples were evaluated in okapi at 116 days or greater of gestation. Analysis of PSPB can be used to augment pregnancy diagnosis in okapi, a species that is of high conservation value and has documented pregnancy-associated morbidity.