We present an integrated microfluidic probe that enables the measurement of multiple kinase activities in selected phenotypically distinct single cells from large cell populations on standard tissue culture platforms. The contents of a cell are captured without disturbing its extracellular context by creating a small lysis zone at the probe tip by hydrodynamic confinement. Pneumatic micro-valves are then used to separate and mix the captured lysate into different assay mixtures in separate small volume chambers for a fluorimetric assay. We demonstrate here the ability to simultaneously measure the activity of three kinases: Akt, MAPKAPK2, PKA and a loading control enzyme, GAPDH, from a single cell. This single cell assay platform enables the correlation of cellular phenotype to intracellular biochemical state at the single cell level and hence can provide a clearer understanding of cell behavior in heterogeneous cell populations.