Background: Cheetahs in captivity are frequently afflicted by chronic disease conditions that are rare in their free-ranging counterparts and in other felids. To date, clear pathophysiological mechanisms for some of these diseases have not been established. Objective: In order to better understand these conditions in this species, we have adopted a systems biology approach to cheetah metabolism, with the hope of generating new hypotheses for future research. Methods: As part of a comprehensive metabolome, we determine the serum (N = 42) and urine (N = 26) amino acid profiles of apparently healthy captive cheetahs, housed at the AfriCat Foundation in Namibia, using GC-MS and LC-MS-MS. Results: A total of 36 serum and 38 urine amino acids were identified and quantified. Glutamine was detected in the serum at the highest mean concentration (1624 µmol/L) followed by alanine (771.9 µmol/L), arginine (669.8 µmol/L) and glycine (478.7 µmol/L). Serum glycine, hydroxyproline, prolylproline, proline and serine concentrations declined significantly with age. Arginine was excreted at the highest mean concentration (740 µmol/L), followed by glutamine (437.1 µmol/L), alanine (397.1 µmol/L) and serine (331.1 µmol/L). The fractional excretion of cystine was by far the highest at 21.92%, followed by hydroxylysine (6.34%), prolinehydroxyproline (5.49%) and α-aminopimelic acid (4.89%). Both urinary glycine and proline-hydroxyproline concentrations decreased significantly with age. None of the serum or urine amino acid concentrations differed significantly between males and females. Conclusion: This study provides some foundational information on the serum and urine amino acid profiles of healthy captive cheetahs.