Schirmer tear test (STT), intraocular pressure (IOP), and ocular biometry were measured in 58 healthy cheetahs (Acinonyx jubatus) between the ages of 2 and 14 yr in two captive groups. During routine health examination under immobilization, each animal underwent ophthalmic examination including STT, IOP, slit-lamp biomicroscopy, indirect ophthalmoscopy, and ocular ultrasonography. Mean ± SD values for STT and IOP were 13.58 ± 5.29 mm/min and 31.40 ± 5.01 mm Hg, respectively. The time between immobilization and examination significantly influenced both STT and IOP measurements. For IOP measurements, a significant group effect was also demonstrated, possibly due to the different anesthetic drug combinations used. Mean ± SD values for biometry were as follows: globe length (32.73 ± 1.74 mm), lens thickness (7.25 ± 1.09 mm), anterior chamber (8.54 ± 1.21 mm), and posterior segment (16.31 ± 2.06 mm). Surprisingly, all of the biometry measurements differed significantly between the two groups. Establishing STT, IOP, and biometric reference values is important for evaluating the ocular health status of the species. These reference values will assist veterinarians working with cheetahs to perform a full and informed ophthalmic examination.