Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of researchers. These studies have provided new information of the cell biology of these parasites and have supported investigators to assume that some of the protozoa form the roots of the evolutionary tree of eukaryotic cells. The cell fractionation usually starts with disruption of the plasma membrane, using conditions that minimize damage to the membranes bounding intracellular organelles. An important requirement for successful cell fractionation is the evaluation of the isolation procedure that can be made by morphological and biochemical methods. The morphological approaches use light and electron microscopy of thin section of different fractions obtained, and the biochemical methods are based on the quantification of marker enzymes or other molecules (for instance, a special type of lipid, an antigen, etc.). Here we will present our experience in the isolation and characterization of some structures found in trypanosomatids and trichomonads.