The cell wall is an external envelope shared by yeasts and filamentous fungi that defines the interface between the microorganism and its environment. It is an extremely complex structure consisting of an elastic framework of microfibrillar polysaccharides (glucans and chitin) that surrounds the plasma membrane and to which a wide array of different proteins, often heavily glycosylated, are anchored in various ways. Intriguingly, these cell wall proteins (CWPs) play a key role in morphogenesis, adhesion, pathogenicity, antigenicity, and as a promising target for antifungal drug design. However, the CWPs are difficult to analyze because of their low abundance, low solubility, hydrophobic nature, extensive glycosylation, covalent attachment to the wall polysaccharide skeleton, and high heterogeneity. We describe a typical procedure of cell wall fractionation to isolate and solubilize different CWP species from yeasts and filamentous fungi according to the type of linkages that they establish with other wall components and under suitable conditions for following reproducible proteomic analyses. CWPs retained noncovalently or by disulfide bonds are first extracted from isolated yeast or fungal cell walls by detergents and reducing agents. Subsequently, CWPs covalently linked to or closely entrapped within the internal glucan-chitin network are sequentially released either by mild alkali conditions or by enzymatic treatments first with glucanases and then with chitinases. This strategy is a powerful tool not only for obtaining an overview of the sophisticated cell wall proteome of yeasts and filamentous fungi, but also for characterizing mechanisms of incorporation, assembly and retention of CWPs into this intricate cellular compartment and their interactions with structural wall polysaccharides.