The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.