Intraocular pressure (IOP) measurement is a common procedure during eye examinations in birds. Differences in the IOP between avian species have been reported, which suggests the need to establish species-specific reference ranges. To determine IOP values of captive black-footed penguins (Spheniscus demersus), we obtained IOP readings with the use of a rebound tonometer by using two established calibration settings (dog and horse). No difference was seen in the IOP between the left and right eye when the horse setting was used; however, a difference was present when using the dog setting. No significant difference between the IOP of male and female penguins was seen in both eyes when the dog or horse setting was used. Rebound tonometry appears to be a safe and repeatable method to obtain IOP values in black-footed penguins.