Basophil activation has been implicated in allergy reaction and immune modulation. Signaling transduction, mediator release and morphology change of basophils have been largely deciphered. This study examined the feasibility of using proteomic strategy to investigate the whole cell proteomic changes in KU812 basophils following activation with phorbol myristate acetate (PMA) plus ionophore (A23187). The two-dimensional electrophoresis (2D) coupled with matrix-assisted laser desorption/ionization-quadrupole/time-of-flight (MALDI-Q-TOF) was selected for studying comparative proteome differences; and cytokine mRNA expressions were monitored. The differentially displayed proteins were further validated by 1D-and 2D-Western blotting for protein expressions. IL-4, IL-5, and IL-13 mRNAs in KU812 were significantly induced; a phenomenon similar to the allergy reaction. The protein expressions of heterogeneous nuclear ribonucleoprotein K (HNRPK) and a-enolase (ENO1) were shown a good correlation according to the results from the 2D, and 2D-Western blots. Since the majority of the identified proteins were associated with immunological, inflammatory disorders and tumor genesis, this study in defining the proteomic changes in basophilic cells that occur in response to treatment with PMA plus A23187 can serve as biomarkers for characterization of in vitro immuno-stimulation.