The Rho-associated kinases (ROCKs) can regulate cell shape and function by modulating the actin cytoskeleton. ROCKs are serine-threonine protein kinases that can phosphorylate adducin, ezrin-radixin-moesin proteins, LIM kinase, and myosin light chain phosphatase. In the cardiovascular system, the RhoA/ROCK pathway has been implicated in angiogenesis, atherosclerosis, cerebral and coronary vasospasm, cerebral ischemia, hypertension, myocardial hypertrophy, and neointima formation after vascular injury. ROCKs consist of two isoforms: ROCK1 and ROCK2. They share overall 65% homology in their amino acid sequence and 92% homology in their amino kinase domains. However, these two isoforms have different subcellular localizations and exert biologically different functions. In particular, ROCK1 appears to be more important for immunological functions, whereas ROCK2 is more important for endothelial and vascular smooth muscle function. Thus, the ability to measure ROCK activity in tissues and cells would be important for understanding mechanisms underlying cardiovascular disease. This chapter describes a method for measuring ROCK activity in peripheral blood, tissues, and cells.