Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC50 values for the prototypic Rho-kinase inhibitors Y-27632 (1.2 +/- 0.05 mu M) and Fasudil (3.7 +/- 1.2 mu M) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC50 value of 77 +/- 30 nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R-2 = 0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC50 values and to screen a larger set of small molecule Rho-kinase inhibitors and Suggests that this approach can be broadly applied to other cell lines and signaling pathways. (C) 2008 Elsevier Inc. All rights reserved.