Cuban tree frogs (Osteopilus septentrionalis) are native to the West Indies and represent one of over 7,000 extant species of amphibians. Biochemical enzymes are often used as an aid in the diagnostic evaluation of many veterinary patients, including amphibians. Knowing the tissue of origin for these enzymes aids in interpretation. This study evaluated tissue specificity of eight different enzymes in the Cuban tree frog, including amylase, lipase, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), and gamma-glutamyl transferase (GGT). Seven different tissues (liver, cardiac muscle, lung, kidney, skeletal muscle, gonad, and pancreas) were collected from five adult Cuban tree frogs for enzyme analysis. The results showed that CK and LDH enzymes were primarily derived from skeletal and cardiac muscles; ALT was primarily derived from kidney and liver; AST was primarily derived from liver, kidney, and skeletal and cardiac muscles; GGT was primarily derived from kidney; ALP was primarily derived from kidney and cardiac muscle; and lipase was primarily derived from liver. Amylase was found to be significantly highest in the plasma and did not have any other tissue specificity. The results of this pilot study show the majority of the tissue enzymes have some tissue specificity and that these results may be of use to clinicians attempting to interpret biochemistry enzyme concentrations from blood samples. However, follow-up studies that correlate blood and tissue concentrations of these enzymes are needed to determine their value.