Olive (Olea europaea L.) pollen is a major health concern in the Mediterranean countries and some olive growing regions in America and Australia. The molecular variability of pollen allergens constitutes a handicap for commercial extract standardization, which is the base of current diagnosis and vaccination procedures. In this paper, we report a time-saving and plant material saving multiplex detection method for the rapid and simultaneous analysis of Ole e 1, Ole e 2, and Ole e 5 allergen polymorphism on a single blot. This method combines high-resolution 2DE techniques with high-sensitive fluorescence-based detection methods. Using this strategy, we were capable to identify a higher number of allergen forms compared with classical 1D approach. The use of fluorescent probes and the increased resolution of 2D blots avoided overlapping effects, and allow estimating the amount of individual allergen forms. In addition, the pattern and identity of the IgE-reactive proteins of either a population or individual patients allergic to olive pollen was also effortlessly determined in a single additional step. This flexible method might be extended to a higher number of olive allergens and cultivars, and is also applicable to other allergogenic plant species and sources.