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. 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. 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. 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%), proline-hydroxyproline (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. This study provides some foundational information on the serum and urine amino acid profiles of healthy captive cheetahs.