AbstractBackgroundOcular hypertension is one of the most underdiagnosed ocular abnormalities among guinea pigs around the world.ObjectivesThe current study investigates the effect of 0.0015% preservative‐free tafluprost ophthalmic solution (Zioptan) on the intraocular pressure of 16 healthy male guinea pigs (Cavia porcellus) under different light/darkness regimes.MethodsAll guinea pigs received a single drop of tafluprost at 5:30 in the right eye, whereas the contralateral eyes served as control to receive a placebo. Then, the animals were randomly divided into two groups; group A was exposed to light, whereas group B was placed in darkness from 5:30 to 18:00. Rebound tonometry (TonoVet) was instrumented to measure IOP values at 5:30 (baseline), 6:00, 7:00, 8:00, 9:00 and then every 3 h until 18:00.ResultsThe maximum IOP reduction associated with tafluprost was observed at 6:00 by −1.4 ± 1.1 mmHg (p‐value = 0.026) and −2.5 ± 1.2 mmHg (p‐value = 0.011) in group A and B, respectively (repeated measure ANOVA test). There was a significant difference between the mean right and left eye IOP values in both groups at 5:30, 6:00, 7:00 and 8:00 (p‐value <0.05), which was greater in amount in group B compared to group A due to the effect of darkness on IOP reduction.ConclusionsIt is suggested that the variations of IOP in different light/dark conditions be taken into consideration when applying ocular hypotensive agents on guinea pigs’ eyes.