We report a technique for detecting protein phosphorylation that involves isoelectric focusing in a vertical mini-gel format followed by immunoblot detection of the target protein. This method uses standard protein gel equipment, allows sensitive detection of protein phosphorylation when phosphospecific antibodies are not available, and provides a stoichiometric measure of phosphorylation. We demonstrate the application of this method for observing phosphorylation of an Arabidopsis thaliana protein in response to biotic stress.