To evaluate the effect of a 6-week washout period on intraocular pressure (IOP) following long-term monotherapy prostaglandin use. Prospective, randomized, controlled, single-centre, single-blinded, parallel-group clinical study. Subjects aged >18 years diagnosed with open-angle glaucoma or open-angle glaucoma suspects based on elevated IOP in one or both eyes, using monotherapy topical latanoprost, bimatoprost, or travoprost once daily. Subjects were prospectively randomized to continue prostaglandin analogue (PGA) monotherapy (control group) or discontinue PGA monotherapy (washout group) for 42 days. IOP was measured at day 0 (day of randomization), 7, 21, and 42. Mean IOP (mm Hg) ± standard deviation. 154 eyes (87 participants) completed the study, with 69 eyes (39 participants) in the control group and 85 eyes (48 participants) in the washout group. In the control group, day 0 IOP (14.64 ± 2.68 mm Hg) did not significantly differ from IOP at days 7 (14.25 ± 3.01 mm Hg), 21 (14.57 ± 2.61 mm Hg), and 42 (14.78 ± 2.30 mm Hg) (all p > 0.30). In the washout group, mean IOP values at days 7 (16.19 ± 3.80 mm Hg), 21 (17.28 ± 3.55 mm Hg), and 42 (17.84 ± 3.31 mm Hg) were significantly greater than those at day 0 (14.48 ± 1.94 mm Hg) and day-matched control group values (all p < 0.002). In the washout group, 24.7% of eyes had a day 42 IOP ≥21 mm Hg. No eyes in the control group had a day 42 IOP ≥21 mm Hg. Six weeks of PGA washout after long-term monotherapy resulted in a small but statistically significant IOP increase. Majority of washout group participants maintained an IOP lower than 21 mm Hg after the 6-week washout duration. (https://clinicaltrials.gov/ identifier, NCT03534882).