Kinases and phosphatases play key roles in the diverse networks transmitting signals within and between cells. Most assays of physiological kinase activation involve measurements representing the average of the response of large populations of cells, which may or may not be heterogeneous in their intensity or time course. Moreover, the preparation of cellular extracts can often introduce inaccuracies into the process. Allbritton and colleagues have now developed a method to assay the activation of individual kinases within single mammalian cells. The method relies on chemical separation to identify and quantify fluorescent peptide substrates that have been injected into individual cells. The electrophoretic mobility of the substrates changes upon phosphorylation, and the latter are easily detected by capillary electrophoresis coupled to laser-induced fluorescence (see pp. 309–312).