The serine/threonine kinases ROCK1 and ROCK2 are direct targets of activated rho GTPases, and aberrant rho/ROCK signaling has been implicated in a number of human diseases. We have developed novel methods for high-throughput assays of ROCK inhibitors that provide for quantitative evaluation of the ability of small molecules to inhibit the function of ROCK kinases in intact cells. Conditions for extraction of known phosphorylated substrates of ROCK were identified, and the involvement of ROCK in phosphorylation of these substrates was evaluated using small interfering RNA (siRNA). Of the potential substrates tested, MYPT1 was identified as a substrate whose phosphorylation was reduced markedly in the combined absence of ROCK1 and ROCK2 proteins, and ELISA methods were developed to allow quantitative measurement of the degree of phosphorylation of MYPT1 at residue T853 in cells grown in 96-well plates. These methods are amenable to high-throughput assays for identification of ROCK inhibitors within libraries of small molecules and can be used to compare compound potencies to prioritize compounds of interest for additional evaluation. These methods should be useful in drug discovery efforts directed toward identifying potent ROCK inhibitors for potential treatment of cancer, hypertension, or other diseases involving rho/ROCK signaling.