Absolute quantification methodologies, which allow the determination of protein concentrations in biological samples such as cell, tissue and body fluid. Recently, protein quantification methodologies mainly are one which rely on the isotope dilution for absolute quantification of proteome and another one which rely on the statistic analysis of MS data called the lable-free technique. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy) or PrESTs (PrESTs-SILAC). Because isotope dilution can provide accurate and presice absolute quantification, so they are crucial for specific applications such as the evaluation of clinical biomarker candidates and understanding the biological function of proteins. In this review, we present a critical overview of these isotope dilution methodologies.