A total of 15 healthy captive black-tailed prairie dogs (Cynomys ludovicianus) of both sexes were studied to determine urine properties of this species as part of an overall clinical health evaluation performed under general isoflurane anesthesia. Each animal underwent a complete physical examination, complete blood count, serum biochemistry panel, whole-body radiology, and cardiographic examination. Urine samples were collected via ultrasound-guided cystocentesis. Each urine sample was evaluated biochemically for the presence of glucose, bilirubin, ketones, pH, proteins, hemoglobin, specific gravity, creatinine, and gamma-glutamyl transferase (GGT); macroscopically or microscopically for color, clarity, erythrocytes, leukocytes, casts, epithelial cells, and crystals; and for pathogenic leptospires by polymerase chain reaction testing. Urinary GGT to creatinine and protein to creatinine ratios were also calculated. Cloudy urine was common but not as a result of calciuria as observed in other rodents. The primary urine properties identified in this study are presented (median: minimum to maximum) and include the specific gravity (1.25: 1.005 to 1.059), GGT (883: 12 to 5833 U/L), creatinine (6064: 1485 to 15,939 μmol/L or 68.6: 16.8 to 180.3 mg/dL), protein (19: 6 to 124 mg/dL), GGT to creatinine ratio (0.112: 0.008 to 0.606), and protein to creatinine ratio (0.3: 0.2 to 1.0), which was higher in males when compared with females. Urine pH as determined by the urine dipstick was 8 to 8.5. New data presented in this report can promote better physiological understanding and improve clinical management of this rodent species.