Recently, many software tools have been developed to perform quantification in LC‐MS analyses. However, most of them are specific to either a quantification strategy (e.g. label‐free or isotopic labelling) or a mass‐spectrometry system (e.g. high or low resolution). In this context, we have developed MassChroQ (Mass Chromatogram Quantification), a versatile software that performs LC‐MS data alignment and peptide quantification by peak area integration on extracted ion chromatograms. MassChroQ is suitable for quantification with or without labelling and is not limited to high‐resolution systems. Peptides of interest (for example all the identified peptides) can be determined automatically, or manually by providing targeted m/z and retention time values. It can handle large experiments that include protein or peptide fractionation (as SDS‐PAGE, 2‐D LC). It is fully configurable. Every processing step is traceable, the produced data are in open standard formats and its modularity allows easy integration into proteomic pipelines. The output results are ready for use in statistical analyses. Evaluation of MassChroQ on complex label‐free data obtained from low and high‐resolution mass spectrometers showed low CVs for technical reproducibility (1.4%) and high coefficients of correlation to protein quantity (0.98). MassChroQ is freely available under the GNU General Public Licence v3.0 at http://pappso.inra.fr/bioinfo/masschroq/.