Leopard sharks (Triakis semifasciata) are temperate, Eastern Pacific elasmobranchs popular in public aquariums. Blood analysis is commonly used for assessing animal health, yet reference values have not been established for this species. This study analyzed T. semifasciata population data to characterize blood reference values for a collection of T. semifasciata housed at a public aquarium. Twenty-one captive leopard sharks were sampled. Blood was collected during annual health examinations from sedated animals. After collection, blood samples were anticoagulated with lithium heparin, and hematocrit and plasma biochemistry values were analyzed. The minimum–maximum ranges were hematocrit 11–31%, buffy coat 1–2%, glucose 4.94–9.38 mM/L, sodium 244–272 mM/L, potassium 3.7–5.5 mM/L, chloride 214–246 mM/L, aspartate aminotransferase 5–31 U/L, creatine kinase 36–1,136 U/L, calcium 3.65–3.95 mM/L, phosphorus 1.13–2.23 mM/L, total protein 21–38 g/L, and total CO₂ 12–18 mM/L. The values identified will contribute to a better understanding of captive leopard shark physiology and to improved veterinary care for captive leopard sharks. Further research can examine the validity of machines like the Vetscan VS2, which will expand the resources available to care professionals.