Subcellular fractionation of Euglena gracilis has been conducted for over 50 years in various forms by numerous research groups. The development of this technique is closely tied to the specific organelle or fraction required for specific purposes. In this chapter, we describe our approach to this process and discuss the insights we gain from it. Sucrose and iodixanol gradients are employed to separate the main organelles of interest; however, these methods alone do not lead to the complete purification of the organelles.