Proteases have been implicated in the tumorigenesis and aggressiveness of a variety of cancer types. In fact, proteases have proven to be very clinically useful as tumor biomarkers in the blood of patients. Proteases are typically involved in complex systems of substrates, activators, and inhibitors, thus making our ability to establish their exact function in cancer more difficult. Trypsin, perhaps the most famous of proteases, has been shown to play a role in cancer progression, but its functional role in ovarian cancer has not been much studied. PAR2, a transmembrane receptor that is known to be activated by trypsin, has been reported to be associated with ovarian cancer. Here, we found that stimulation of ovarian cancer cell lines with trypsin or PAR2 activating peptide markedly increased MAPK signaling and cell proliferation. Additionally, HE4, a WAP-family glycoprotein and ovarian cancer biomarker, was found to inhibit trypsin degradation, thereby retaining its activity. Patient data seemed to support this phenomenon, as the serum of ovarian cancer patients with high HE4 expression, revealed significantly elevated trypsin levels. These data support the hypothesis that trypsin plays a tumorigenic role in ovarian cancer, which can be mediated by its receptor PAR2, and potentiated by HE4.