New advances in mass spectrometry (MS) have pushed the limits of sensitivity, enabling comprehensive characterization and accurate quantitation of complete proteomes.  Despite rapid advances in analytical instrumentation and software, data quality to a large extent still depends on upstream sample preparation techniques. A significant reduction in variability should be possible by standardizing sample preparation steps and analyzing accurate quantities of peptide material in each LC/MS analysis.  In this study, we utilized a recently introduced peptide quantitation assay to monitor the protein and peptide concentration at each step of Thermo Scientific tandem mass tag^TM (TMT^TM) reagents sample preparation workflow.  Standardizing the digestion, labeling and fractionation steps using  a peptide quantitation assay significantly improved reproducibility and quality of the MS results.