The effects on the protein pattern of raffinose family oligosaccharides ethanol extraction from intact lupin seeds (Lupinus albus and Lupinus luteus) has not been addressed so far. In this work, 1D and 2D electrophoretic techniques were used to detect changes of the protein profiles upon oligosaccharide removal procedure. Some differences between untreated and treated samples were clearly visible in 1D electrophoresis, where the decrease of some polypeptides was revealed. In addition, a dark zone at the top of each treated sample lane became visible, suggesting the formation of large-sized protein aggregates as a result of the extraction procedure. By using the greater resolution of 2D electrophoresis, the identification of some varying spots was made possible: In particular the bands corresponding to conglutin γ were significantly reduced in the processed samples. The leakage of this protein by ethanol treatment of the seeds was qualitatively and quantitatively confirmed with specific antibodies by Western and dot blotting techniques, respectively. Another polypeptide around 100 kDa undergoing decrease upon treatment was tentatively identified as lupin lipoxygenase, according to literature data and direct lipoxygenase activity measurements on treated and untreated seed extracts. The observed decrease of lipoxygenase activity in the processed seeds was 37%. These results show that the extraction of α-galactosides, while maintaining the overall pattern of lupin storage proteins, led to the reduction of some critical proteins. In particular, conglutin γ and lipoxygenase decrease could be desirable in view of their potential allergenicity and effects on the flour organoleptic characteristics, respectively.