Despite the popularity of leopard geckos (Eublepharis macularius) in the pet trade, packed-cell-volume (PCV) and biochemistry reference intervals are still lacking for these animals. The risk of inducing tail autotomy during venipuncture is of particular concern in this species. Blood samples were obtained from 26 healthy subadults (14 females and 12 males) from the ventral coccygeal vein or the cranial vena cava under general anesthesia with isoflurane. Plasma biochemistry panels were performed by a reference laboratory and PCV was measured. Reference intervals were established following the American Society for Veterinary Clinical Pathology guidelines. Reference values were established for parameters with less than 20 available results because of limited sample volumes. Results were computed using the Reference Value Advisor software. Venipuncture was performed safely, without tail autotomy, in all 26 individuals. No lymph contamination was detected macroscopically. Reference intervals were 21–40% for PCV, 33–394 µmol/L for uric acid (0.5 to 6.6 mg/dL), 0–3,701 IU/L for creatine kinase, and 0.6–37.5 µmol/L for bile acids. Reference values were established for the following parameters: 0.5–5.3 mmol/L for phosphorus (1.5–16.4 mg/dL), 24–80 g/L for total protein (2.4–8.0 g/dL), 3.5 to >9.25 mmol/L for total calcium in females (14 to >37 mg/dL), and 11–65 IU/L for aspartate aminotransferase. Biochemistry results in leopard geckos were similar to those reported in crested geckos (Rhacodactylus ciliatus).