Given that nonadherence is related to subject characteristics and drug tolerance and preserved eye drops tend to be more intolerable than preservative-free ones, we conducted a phase 4, parallel-grouped, investigator-blind, active-control, randomized, multicenter study. A total of 51 patients with intraocular pressure (IOP) ≥ 15 mmHg diagnosed with open-angle glaucoma or ocular hypertension were randomly assigned to the preserved latanoprost group (n = 26) and the preservative-free latanoprost group (n = 25). The efficacy variables were corneal/conjunctival staining grade, Ocular Surface Disease Index (OSDI), adherence at 12 weeks after the first administration; corneal/conjunctival staining grade at 4 weeks; and IOP, tear break-up time (TBUT), and hyperemia score at 4 and 12 weeks. The safety variables included visual acuity and drug tolerance questionnaire results. There was no statistically significant difference in corneal/conjunctival staining grade, OSDI, or TBUT between the groups at 4 and 12 weeks. However, the adherence rate was higher and the hyperemia score was lower in the preservative-free group than in the preserved group. The severity and duration of stinging/burning sensation were lower in the preservative-free group than in the preserved group. Overall, preservative-free latanoprost showed better ocular tolerance assessed by hyperemia scores and stinging/burning symptoms following higher adherence than preserved latanoprost.