Gastrointestinal disease is a common clinical problem in captive cheetahs (Acinonyx jubatus). It is reported that gastritis affects the vast majority of the captive population of cheetahs. Pancreatitis and acute and chronic enteritis have also been reported. These issues pose significant long-term health and welfare implications for cheetahs. Cobalamin, folate, methylmalonic acid (MMA), gastrin, feline pancreatic-specific lipase immunoreactivity (fPLI), and feline trypsin-like immunoreactivity (fTLI) immunoassays are important biomarkers of gastrointestinal disease in domestic cats. The goal of this study was to determine if these immunoassays validated in domestic cats could be used clinically in cheetahs, by establishing reference intervals (RI) for these biomarkers in cheetahs. A cohort of 40 clinically healthy cheetahs was selected from three zoological institutions on the basis of being free of clinical gastrointestinal disease and extra-gastrointestinal disease that could affect biomarkers, as well as having banked frozen serum. Cheetah biomarker RI, with domestic cat RI for comparison in parentheses, are as follows: cobalamin 470–618 pg/ml (290–1500 pg/ml), folate 2.2–15.7 ng/ml (9.7–21.6 ng/ml), MMA 365–450 nM/L (139–897 nM/L), fPLI 0.5–1.2 µg/L (0–4 µg/L), and gastrin 30–50 pg/ml (<10–39.5 pg/ml). This study shows that RI for gastrointestinal biomarkers can be notably different, even between species that are as closely related as the domestic cat and the cheetah. Additionally, it was found that the fTLI assay does not cross-immunoreact with cheetahs. In conclusion, this study emphasizes the importance of developing species-specific RI for biomarker assays and using caution when extrapolating RI from other species.