Abstract: Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only approximately 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of approximately 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), approximately 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.