Venom immunotherapy (VIT) is an adequate model to explore the immune mechanisms underlying this type of treatment. We have investigated the use of protein arrays to detect variations in the levels of cytokines in patients receiving VIT. In the present study we selected 11 non-atopic patients with systemic reactions after Hymenoptera sting that received VIT during at least three years. In order to evaluate the success of VIT all of them should have tolerated a sting field after VIT. Serum samples were obtained before initiating VIT and after at least three years of successful VIT. We analyzed 42 serum proteins corresponding to a Th1/Th2 panel using protein array methodology. We observed a significant increase of Interleukin 10, Myeloid Macrophage Colony Stimulation Factor, Macrophage Derived Chemokine, Interleukin 1-, Vascular Endothelial Growing Factor and Stem Cell Factor serum levels after successful VIT. We discuss the usefulness and normalization of this array method to analyze cytokines and other serum proteins. Monitoring these serum cytokines could help to predict the response and to elucidate the mechanisms underlying immunotherapy.