The inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. Characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. Demembranated Xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and to control the cell cycle state of the egg extract. This system has become an invaluable and widely used tool for studies of cell cycle regulation and many downstream events. Here, we describe a protocol, derived in part from other published protocols and modified over time, for the preparation of Xenopus sperm nuclei that can be used in a variety of in vitro assays.