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Comprehensive proteome analysis requires efficient, robust, and practical methods for the identification and quantification of all proteins in complex biological samples. Mass spectrometry is a well‐established protein identification tool and semiquantitative proteomics has traditionally been performed using two‐dimensional gel electrophoresis (2‐DE) coupled with mass spectrometry. However, other separation strategies based on liquid chromatography now complement 2‐DE as a central method for analysis of proteins in complex biological systems. In this review, we discuss the most promising methodological developments in this area with particular attention to their merits and limitations.