Mass-spectrometry-based proteomic approaches are increasingly applied to biological and clinical studies. Initially used by specialized laboratories, the technology has matured and gained acceptance by the community, using various analytical processes and platforms. To facilitate data comparison and integration across laboratories, there is a need to harmonize analytical processes to ensure the generation of reliable proteomic data sets. This is especially critical in the context of large initiatives, such as the Human Proteome Project promoted by the Human Proteome Organization (HUPO). Quality control is a first step toward the harmonization of proteomics data sets. We have developed a procedure to routinely assess the uniformity of proteomics analyses. It relies on a simple protocol based on three proteins and two sets of isotopically labeled peptides, one being added prior to tryptic digestion and the second one prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The proposed method evaluates in a single step both the sample preparation, by measuring the relative amounts of endogenous peptides and their isotopically labeled counterparts, and the LC-MS platform performance, by monitoring the main LC-MS attributes for reference peptides. The procedure is simple and easy to implement into routine workflows typically employed by the proteomics community.