Methods and reagents are provided for determining the activation state of a signal transduction pathway signaling protein. There exists a need in the art for methods that can monitor the efficacy of a signal transduction inhibitor in a patient. Other needs exist for detecting and monitoring certain disease or disorders that are associated with aberrant activation of a signal transduction pathway signaling protein. The present assay provides a highly sensitive assay that is also useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates.