Yeast cells display cell walls that must first be broken before the addition of detergents for lysis. This method describes the use of glass beads in combination with a mechanical bead beater to disrupt cell walls of both  Saccharomyces cerevisiae  or  Schizosaccharomyces pombe  directly in a nonionic detergent Lysis buffer containing 0.1% Nonidet P-40. Alternatively, this protocol can be applied for the lysis of yeast cells in Lysis buffer without detergent; upon completion of the bead beating, Triton X-100 is added to complete lysis. Yeast cells are cultured and collected while in log phase before being washed once and mixed together with glass beads in a tube. The applied shaking process facilitates disruption of the cell walls, upon which separation of yeast and glass beads is accomplished by forcing lysed cells through a hole created in the bottom of the tube during the centrifugation process. An alternative bead-beating protocol details the use of Lysis Buffer 2, which does not contain detergents and calls for the addition of Triton X-100 after cell lysis in the presence of glass beads. Use of Lysis Buffer 2 without detergent may avoid bubble and foam formation during the bead-beating process that could potentially denature proteins.