Xenopus laevis oocytes are popular cells in experimental biology. Fully grown oocytes are large (∼1.3-mm diameter) with an enormous nucleus (∼300-µm diameter). Oocytes are generally isolated by either manual dissection (manual defolliculation) or enzymatic (mainly with collagenase preparations) digestion of the extracellular connective tissues. In this chapter, we describe both procedures, which are routinely used in our laboratory. However, manual defolliculation does not actually remove the innermost layer of follicle cells, which are anchored to the vitelline membrane. To remove these follicle cells, further mechanical or enzymatic treatment is required. On the other hand, many have experienced nonspecific effects with collagenase-treated oocytes, including spontaneous oocyte maturation and reduced oocyte health. We discuss possible explanations and solutions to these problems. Finally, we also describe procedures we employ routinely to isolate oocyte nuclei and enucleated oocytes.