We report a protocol for pre-fractionation of proteins from frozen tumours. Using this protocol two separate protein fractions are extracted: Fraction 1 is enriched for cytosolic and Fraction 2 for nuclear/nuclear membrane proteins. The accuracy and reproducibility of the protocol were demonstrated by Western blot analyses and SELDI-TOF-MS profilings. The resulting protein profiles were similar within the respective groups of Fractions 1 and 2 samples, without differences between freshly prepared tumours or those obtained as frozen tissue from biobanks.