Mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) family are activated in response to many growth and differentiation factors as well as some oncogenes. ERK activation follows phosphorylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exemplified by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target substrates which include stathmin, a phosphoprotein regulating microtubule stability. We report here the development of a simple, 96-well plate, quantitativein vitroassay measuring purified ERK2 catalytic activation by a constitutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by33P phosphorylation of purified biotinylated stathmin captured on streptavidin-coated scintillation proximity assay beads which eliminates the need for wash steps. The assay was optimized and theK0.5value for ATP was found to be 0.9 μM and theKmfor stathmin was determined to be 16 μM. The assay was also used to determine IC50values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantitative measurements of MEK1-dependent ERK2 activation to be performed in a day.