Cancer is a disease which develops as the result of many negative changes in the biology of otherwise healthy tissue. Non-invasive imaging of aberrant, tumor-associated molecules or processes can be used to isolate and characterize the malignancy, but first one must identify those characteristics which differentiate normal and cancerous tissues. Proteases are associated with cancer growth and progression and are often considered in the search for biomarkers to be used in tumor detection and evaluation. Membrane-Type Serine Protease 1 (MT-SP1) is a membrane anchored protease which is upregulated in epithelial cancers. A dysregulation in MT-SP1/matriptase levels with respect to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 [HAI-1] suggests that it is an increase in proteolytic activity that significantly differentiates malignant from normal tissue. The Craik lab has developed antibodies which bind to and inhibit MT-SP1, but the utility of these probes, and of MT-SP1 activity as a cancer-associated biomarker, has yet to be established. Here we show that these antibodies inhibit the full-length protein on the surface of cancer cells and that they may be functionalized for imaging of this activity in vivo. The antibodies were first used to target and inhibit MT-SP1 on human cancer cell lines which express MT-SP1 mRNA. This activity was then targeted in vivo using xenograft mouse models of cancer. It was found that the antibodies preferentially localize to tumors which express MT-SP1, suggesting that MT-SP1 activity is a novel biomarker for epithelial cancer and these antibodies provide a non-invasive method for detecting this activity in vivo.