The aim of this study was to evaluate the effects of 0.0015% preservative-free (PF) tafluprost alone and in combination with 0.5% timolol maleate or0.5% betaxolol HCl on Schirmer tear test (STT), intraocular pressure (IOP), and pupil diameter (PD) in clinically normal dogs. Twenty-one healthy adult castrated male cross-bred dogs were used in this study. Dogs were randomly divided into three groups. The first group received one drop of (PF) tafluprost (Taf), in a randomly selected eye. The second group received one drop of (PF) tafluprost plus one drop of timolol maleate (Taf-Tim), and the last group received one drop of (PF) tafluprost plus one drop of betaxolol HCl, (Taf-Bet). In all groups, the fellow eyes were served as control and received one drop of saline as a placebo. IOP, STT, and PD measurements were performed at the baseline and every 30min for the first 2h, every 2 h for the next 10 h, and at 24h and 36h post-instillation (PI). In all groups, significant differences in IOP values were observed between treated and untreated eyes (Taf: p < 0.001, Taf-Tim: p = 0.014, Taf-Bet: p = 0.008). The maximum reduction in mean IOP after unilateral administration of Taf, Taf-Tim, and Taf-Bet was 8.3mmHg, 10.7mmHg, and 13mmHg, respectively. No significant differences in STT values were observed between treated and untreated eyes at any time points. In all groups, significant differences in PD values were observed between treated and untreated eyes in all time points except the baseline and 36h post-drug instillation (p < 0.001). Tafluprost alone or in combination with timolol and betaxolol was able to reduce intraocular pressure. The greatest effect of the drugs occurred 6 and 8h PI. The present study revealed that the combination of tafluprost/betaxolol is more potent in decreasing IOP than tafluprost alone or a combination of tafluprost/timolol in healthy dogs.