Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, while 82 spots in 2D. The corresponding protein bands and spots were excised from the silver stained gel and protein assignation was made by MALDI-TOF/TOF. A total of eleven proteins (including isoforms) were unambiguosly identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin.