One of the major challenges in functional proteomics is the separation of complex protein mixtures to allow detection of low abundance proteins and provide for reliable quantitative and qualitative analysis of proteins impacted by environmental parameters. Prerequisites for the success of such analyses are standardized and reproducible operating procedures for sample preparation prior to protein separation. Due to the complexity of total proteomes, especially of eukaryotic proteomes, and the divergence of protein properties, it is often beneficial to prepare standardized partial proteomes of a given organism to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis. In this chapter we will describe with detailed recipes procedures for the enrichment and isolation of the currently most investigated organelles and subcellular compartments in mammalian cells using classical centrifugation techniques to more sophisticated immunoaffinity-based procedures.