Proteome research aims to unravel the biological complexity encoded by the genome. Due to the complexity of higher eukaryotic cells, single-step characterization of a proteome is likely to be difficult to achieve. However, advantage can be taken of the macromolecular architecture of a cell, e.g., subcellular compartments, organelles, macromolecular structures and multiprotein complexes, to establish subcellular proteomes. This review highlights recent developments in this area of proteomics, namely the establishment of two-dimensional electrophoresis (2-DE) reference maps of subcellular compartments and organelles as well as the characterization of macromolecular structures and multiprotein complexes using a proteomics approach.