Purpose: to determine the main characteristics of the mydriatic effect of drug formulations of phenylephrine, containing and not containing hypromellose, hyaluronic acid as an auxiliary component, in a comparative in vivo study, as well as to study their local bioavailability and the effect on the mucous membrane of the eye. Materials and methods. The studies were carried out on 40 adult male Soviet Chinchilla rabbits. The research model involved the mydriatic impact and the local irritant effect of the drug when instilled into the awake rabbit’s conjunctival sac. The peak concentration of phenylephrine, which is part of all formulations studied, was determined in the aqueous humor of the anterior chamber of the animal's eye at the 5-minute point by high-performance liquid chromatography with tandem mass spectrometric detection. Results. A single instillation of 1 drop of a 2.5% solution of phenylephrine hydrochloride containing hypromellose as an excipient is superior in depth, rate of reaching the maximum mydriatic effect and its duration to the formulation that does not contain hypromellose as an excipient. Only in a few cases, drug formulations containing hypromellose caused an insignificant weak reaction of eyelid closure, in contrast to a moderate local irritant reaction of the rabbit's eye occurring when phenylephrine solution without hypromellose is instilled. Peak concentrations of phenylephrine in the aqueous humor of the anterior chamber of the eye determined 5 minutes after instillation of 1 drop of 2.5% phenylephrine solutions containing hypromellose significantly exceed those occurring when phenylephrine drugs of the same concentration that do not contain hypromellose are instilled. Conclusion. The inclusion of hypromellose as an auxiliary substance into the formulation of phenylephrine eye drops optimizes the pharmacodynamics and pharmacokinetics of the active substance due to faster penetration into the eye anterior chamber aqueous humor, increasing local bioavailability and prolonging the exposure time. In addition, the absence of a local irritating effect of such forms on the eye tissues may also be explained by the presence of hypromellose.