Introduction: Based on the chemical structures of known Rho-kinase-2 inhibitors, 18 benzimidazole derivatives were synthesized and studied for their ophthalmic hypotensive activity in animals with normal intraocular pressure. The dependence of the pharmacological effect on the chemical structure of the compounds was analyzed. The effect of the most active compounds on Rho-kinase-2 activity was assessed. Materials and Methods: Ophthalmic hypotensive activity was assessed by measuring intraocular pressure with a TonoVet veterinary tonometer in 120 mongrel rats (6 in each group) before and after instillation of reference drug solutions ( timolol and melatonin ) and 18 test substances. The effect of the test compounds on Rho-kinase activity was assessed using an in vitro enzyme-linked immunosorbent assay spectrophotometrically. Results: The most active compounds among the new benzimidazole derivatives after a single instillation at a concentration of 0.4% were compound 7a (1-(4-fluorobenzyl)-3-(2-(pyrrolidin-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-imine hydrochloride), which reduced intraocular pressure in normotensive animals by 28.21%, exceeding the effect of the reference drug timolol (26.84%), but inferior to melatonin (30.95%), and compound 1d (1-(1-(2-(azepan-1-yl)ethyl)-1H-benzo[d]imidazol-2-yl)-3-(3-trifluoromethyl)phenyl)urea hydrochloride), which reduced ophthalmotonus by 23.96%, slightly inferior to timolol . The test compounds do not affect intraocular pressure dynamics in the contralateral eye and, therefore, do not have a systemic effect, unlike the reference drugs. It was also found that compounds 7a and 1d at a concentration of 1*10-4 mol/L inhibit Rho-kinase-2 by 26.72% and 18.11%, respectively. Conclusion: The most active compounds, 7a and 1d, were identified as Rho kinase-2 inhibitors that exhibit ocular hypotensive effects in normotensive animals in vivo.