In comparative proteomic studies, it is important to know the variability associated with sample preparation. In this study, we report the strategy of using SILAC (stable isotope labeling by amino acids in cell culture) to evaluate the effect of the variation in sample preparation for quantitative proteomics. Variability can be measured when equal amounts of light and heavy SILAC samples undergo the same sample preparation procedures in parallel, and the two samples are mixed for relative protein quantitation by mass spectrometry. The high quantitative accuracy of SILAC allows for characterization of small variations. First, the reproducibility of immunoprecipitation (IP) and in-gel digestion was evaluated, and the impact of replicate number on quantitative accuracy was characterized. Second, we evaluated the overall variation in a comparative workflow involving three sequential sample preparation steps: IP, SDS-PAGE fractionation, and in-gel digestion. The evaluation of individual sample preparation steps was very valuable for experimental design: the optimal number of replicates for each step could be readily determined and the overall variation of the workflow could be predicted from the variation of the individual steps involved. By using informed experimental design, we demonstrated that the error associated with multiple steps of sample preparation in a comparative experiment can be limited to a reasonably low level.