The successful combination of highly sensitive mass spectrometry and pre-fractionation techniques has provided a powerful tool to detect dynamic changes in low abundant regulatory proteins at the organelle level. Subcellular fractionation, being flexible, adjustable (both in cell and tissues), and allowing the analysis of proteins in their physiologic/intracellular context, has become the most commonly used preparative/enrichment method. This chapter introduces state-of-the-art subcellular fractionation protocols and briefly discuss their suitability, advantages, and limitations.