Filamentous fungi have been recognized as extraordinary producers of secreted proteins and are known to produce novel proteins and enzymes through dispensable metabolic pathways. Here, methods are described for the isolation and enrichment of samples of secreted proteins from cultures of filamentous fungi for analysis by gel electrophoresis and mass spectrometry techniques. These methods can be readily applied to the study of differential protein expression and secretion and metabolic pathways in filamentous fungi by proteomic approaches.