Background and objectives: Bilastine is a non-sedating second-generation antihistamine for the symptomatic treatment of allergic rhinoconjunctivitis and urticaria. This trial aimed to evaluate the optimal dose, efficacy, and safety of a newly developed once-daily preservative-free ophthalmic formulation of bilastine for allergic conjunctivitis. Methods: This phase 2, single-center, double-masked, randomized study evaluated the efficacy of 3 doses of a bilastine ophthalmic formulation (0.2%, 0.4%, and 0.6%) compared to vehicle for the treatment of allergic conjunctivitis. The primary efficacy endpoint was ocular itching reduction. The Ora-CAC® Conjunctival Allergen Challenge model was used to assess ocular and nasal symptoms at the onset of action (15 minutes), 8- and 16-hours post-treatment. Tolerance and safety were also evaluated. Results: A total of 121 adults with seasonal and/or perennial ocular allergy were randomized. Bilastine ophthalmic formulation 0.2%, 0.4% and 0.6% were significantly superior (p>0.001) to vehicle for the treatment of ocular itching at 3, 5 and 7 minutes post-challenge at onset of action (15 minutes) and 8 hours post-treatment. Bilastine 0.6% was also effective at 16 hours post-treatment. Treatment differences for bilastine 0.6% were statistically significant (p<0.001) compared to vehicle at all timepoints for tearing, eyelid swelling, and nasal symptoms. No relevant adverse events were observed. Conclusions: All the tested ophthalmic bilastine doses were efficacious in rapidly reducing ocular itching. The 0.6% formulation was effective up to 16 hours post-treatment, making it suitable for once-daily administration. The new formulation was safe and well tolerated.