Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles-proteomic analysis is discussed as well.