Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.