Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that proteolyze extracellular matrix components as well as a variety of functional proteins. Here we describe a "degradomics" method that efficiently identifies substrates of MMP-14 in a complex protein mixture, such as plasma. Plasma proteins were incubated in the presence or absence of the MMP-14 catalytic domain and displayed on two-dimensional (2-D) gels. After a comparison of the gels, we selected 40 protein spots that reproducibly showed disparities. Upon in-gel digestion, mass determination, and peptide mass fingerprinting, we identified 15 different proteins from 31 spots. These proteins included six known substrates and nine potential substrates of MMP-14. Among the latter, the purified forms of apolipoprotein A-I, apolipoprotein E, and plasma gelsolin were cleaved in vitro by MMP-14, confirming that each of them is a novel substrate of MMP-14. These results demonstrate that our method rapidly and selectively identifies MMP-14 substrates from human plasma proteins. This method would thus constitute a powerful tool for identifying the substrates of MMPs and other proteases in highly complex mixtures of proteins and would enhance our understanding of the biological roles of these enzymes.