Severe allergic ocular diseases as atopic keratoconjunctivitis can induce corneal damage due to inflammatory substances released from giant papillae. Tacrolimus eye drops are one of the current therapeutic alternatives for its treatment. This work is aimed at developing and characterizing a 0.03% tacrolimus ophthalmic formulation, which was introduced in three types of vehicles (BBS, PVA and Hyaluronic Acid). For this, we have performed in vitro (stability studies) and in vivo assays (corneal permanence time measured directly by Positron Emission Tomography) of three potential formulations. Next, the best formulation was selected, and its toxicological profile and clinical effectiveness have been evaluated. The biopermanence studies (direct measurements and PET/CT) showed that the formulations with PVA and Hyaluronic Acid present more retention time on the ocular surface of rats than PBS. From the stability study, we have determined that tacrolimus with PVA in cold storage is the best option. Tacrolimus with PVA has shown lower cytotoxicity than cyclosporine at early times. On the other hand, the pilot study performed has shown significant improvements in patients, with no noticeable adverse reactions. Based on stability, biopermanence, safety and clinical effectiveness studies, we concluded that tacrolimus-PVA eye drops are a suitable candidate for its clinical application in inflammatory ophthalmology diseases.