Ovarian cancer is the leading cause of death among gynecological cancers in the United States. Ovarian cancer employs a unique mode of metastasis, as tumor cells disseminate within the peritoneal cavity, colonizing in several sites and driving the accumulation of ascites. Tumor recurrence and metastasis are significant factors contributing to poor prognosis. The membrane-anchored serine protease testisin is aberrantly expressed in ovarian tumors and it’s only known substrate, protease activated receptor-2 (PAR-2), is also overexpressed in ovarian tumors. In this study I have examined 1) the role of testisin and PAR-2 signaling in ovarian tumor metastasis and 2) determined whether testisin and other membrane-anchored serine proteases may be anti-cancer therapeutic targets. We generated human ovarian ES-2 tumor lines that express testisin or the catalytically inactive testisin mutant S238A and explored the role of constitutive testisin expression in late stage ovarian cancer. We show that testisin stimulates the activation and internalization of PAR-2 resulting in decreased expression of ANG2 and ANGPTL4. Using a preclinical xenograft model of late stage ovarian cancer, we find that testisin activity in ovarian cancer cells reduces intra-peritoneal tumor dissemination, tumor burden and ascites formation. Analyses of the tumors showed that testisin activity downregulates the expression of ANG2 and ANGPTL4 in vivo as well as in vitro. To examine membrane-anchored serine proteases as therapeutic targets in cancer, we utilized an engineered anthrax toxin pro-drug strategy requiring proteolytic cleavage of a protective antigen (PrAg) for activation. We explored the efficacy of these engineered PrAg toxins in several intraperitoneal models of ovarian cancer metastasis and show PrAg treatment reduced tumor burden in both an intraperitoneal NCI/ADR-Res xenograft and an ES-2 minimal residual disease model, which replicates debulking surgery in ovarian cancer patients, but had no effect in a syngeneic ID-8-Luc xenograft model of ovarian cancer. Additionally, we found that engineered PrAg toxins are capable of inducing lung and pancreatic cancer cellular death in vitro. Data presented herein provide new insight into the potential role of testisin and PAR-2 signaling in ovarian cancer metastasis and suggests membrane-anchored serine proteases as a potential therapeutic target for metastatic ovarian cancer.