A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease.