We describe a simple, plate-based method to analyze the secretomes of microorganisms growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The method described here enables rapid large-scale comparative studies of the secretomes of filamentous fungi and other microorganisms growing on a variety of solid substrates, which eventually may have implications for our understanding of enzymatic lignocellulose degradation.