Functional proteome analysis is not restricted to the sequence information but includes the broad spectrum of structural modifications and quantitative changes of proteins to which they are subjected in different tissues and cell organelles and during the development of an organism. Cell biology has provided the means required for the analysis of the composition and properties of purified cellular elements. Subcellular fractionation is an approach universal across all cell types and tissues, including cardiac and vascular system. Subcellular fractionation and proteomics form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach resulting in reduced sample complexity and is most efficiently combined with high-resolution 2D gel/mass spectrometry analysis as well as with gel-independent techniques. In this study we introduce state of the art subcellular fractionation techniques and discuss their suitability, advantages, and limitations for proteomics research.