The yeast cell wall is a dynamic and complex matrix of polysaccharides (glucans, mannans, and chitin), proteins and minor amounts of lipids that isolate the yeast from the extracellular medium, protecting it against osmotic and physical injuries. Removal of this essential structure for cell integrity and viability by controlled enzymatic digestion in an iso-osmotic medium brings about protoplast formation. When yeast protoplasts are incubated in an osmotically stabilized liquid nutrient medium, cell wall precursors are secreted into the culture medium to de novosynthesize the cell wall. During the early stages of the regeneration process of protoplast walls, many wall protein precursors (presumably structural proteins along with remodeling and cross-linking enzymes) are shed into the extracellular medium but not covalently incorporated into the nascent cell wall, intriguingly enabling their easy isolation and solubilization. We have developed a method to collect proteins secreted from yeast protoplasts in active cell wall regeneration under conditions that are suitable for subsequent proteomic analyses. This procedure circumvents some of the troubles intrinsically related to other extraction protocols of cell wall proteins, such as chemical or enzymatic modifications, and poor quality in protein resolution and identification because of linkages to glucan/chitin residues. It further offers a valuable model system to understand how the de novocell wall biosynthesis occurs in the yeast cell or how the yeast cell wall participates in morphogenesis.