RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascu-lar disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibi-tors as potential anti-glaucoma agents. These compounds showed potent IC50 values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (~7 mmHg).22 To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo phar-macokinetic properties were discovered.