Isotachophoresis, with its inherent high resolving power is a rapid and powerful technique for characterization of Hymenoptera insect venoms. By applying a combination of isotachophoresis for the first dimension separation step, and enzyme based assays, electrophoresis or immunological assays for the second dimension identification, it has been possible to identify the most important peptide/protein components in “Yellow Jacket” (Vespula species) venom. The combined two dimensional procedure is rapid, reproducible and well suited for determination of the antigenic/allergenic composition of various Hymenoptera venoms.