Recent studies conducted in our laboratory demonstrate that Aspergillus parasiticussynthesizes and stores aflatoxin in transport vesicles and endosomes. Proteomics data suggest that enzymes involved in the synthesis of other secondary metabolites as well as enzymes involved in response to heat, osmotic, and oxidative stress also localize to these subcellular organelles. In order to better understand how cells integrate the regulation and function of secondary metabolite biosynthesis and stress response, it is important to understand the composition and function of the membrane-bound organelles that house this biosynthetic machinery. Isolation of vesicles, endosomes, and vacuoles (V fraction) is, therefore, an essential method to study secondary metabolism in A. parasiticus at the cellular level. Here, we describe a “one-step density gradient” method for purification of a highly heterogeneous cell fraction consisting of transport vesicles, endosomes, and vacuoles from protoplasts prepared from A. parasiticus cells harvested during aflatoxin synthesis.