The characterization of post translational modified proteins separated by electrophoresis requires the extraction of the intact protein from the gel and removal of other low molecular weight contaminants, such as SDS and salt for an accurate determination of molecular mass.  We evaluated a passive elution method from SDS-PAGE for low molecular mass intact protein followed by molecular mass determination with MALDI-TOF. Ribonuclease was used as a model to evaluate the efficiency of the process. We examined the effect of the time, temperature and quantity of protein on the elution process. SDS-PAGE was carried out with 0.5 to 5.0 μg of Ribonuclease in 12.5% polyacrilamide gels and stained with colloidal Coomassie Brilliant Blue G-250 for 24 hours. The band containing the proteins was excised and destained with a 25% methanol overnight, macerated and incubated in 30 μl 0.1M sodium acetate pH 8.2, overnight at room temperature. Under the conditions employed the percentages of ribonuclease recovery obtained was 20% to 56%, determined by the Lowry method, using bovine albumine as standard. Comassie Brilliant Blue, SDS and salts were removed from the protein sample after passive elution using a Zip-Tip_HPLC (Hidrophobic Reverse Phase Interaction Cromatography) and the proteins were eluted with 60% methanol and 5% formic acid. MALDI-TOF mass spectrometry was carried out using sinapinic acid as matrix. The molecular mass of the eluted protein was compared and matched with the theoretical mass obtained for bovine ribonuclease at the Swiss Prot Data Bank. The methodology described here could applied to study proteins separated by 2D-electrophoresis in order to identify post translational modified.