AIM: To evaluate whether latanoprost/timolol fixed combination (LTFC) dosed twice daily may provide further intraocular pressure (IOP) reduction and evaluate the safety profile at this dose. METHODS: This is an open-labeled, randomized, prospective crossover study on fourty primary open angle glaucoma patients. Two weeks of washout period were followed by randomization to either once daily (OD, group A) or twice daily dosing (BD, group B) of LTFC for 4wk. After another 2-week washout period, the patients’ treatment dose was crossed-over for another 4wk. IOP reduction alongside ocular and systemic side effects were evaluated. RESULTS: Mean baseline IOP was 18.57±2.93 and 17.8±3.01 mm Hg before OD and BD dose respectively, (P=0.27). Mean IOP after BD dose was statistically lower (12.49±1.59 mm Hg) compared to OD (13.48±1.81 mm Hg, P=0.017). Although IOP reduction after BD dose was more (5.32±3.24 mm Hg, 29.89%) than after OD dosing (5.04 mm Hg, 27.14%), it did not reach statistical significance (P=0.68). Patients switched from OD to BD (group A) showed mean IOP reduction by 0.69 mm Hg [95% confidence interval (CI): -0.09 to 1.48 mm Hg, P=0.078]; but patients switched from BD to OD (group B) had significantly higher mean IOP by 1.25 mm Hg (95%CI: -2.04 to -0.46 mm Hg, P=0.006). BD dose had more ocular side effects albeit mild. CONCLUSION: Mean IOP after LTFC dosed twice daily is statistically lower, with additional mild side effects.