Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells and tumor cells in response to chemotherapy, irradiation and oxidative stresses in the tumor microenvironment. Binding of antibodies targeting PS on the tumor endothelial cells and tumors recruit immune cells and engage the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric anti-PS antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in several randomized Phase II trials. In the present study, we demonstrate in vivo tumor imaging of PS expression using real-time, near infrared fluorescence imaging of antibodies that specifically target PS. Two human tumor xenograft models, orthotopic PC-3 prostate tumors in SCID mice and BT474 breast tumors in nude mice, were used to image tumors using a near infrared dye-labeled (NIR) PGN635 F(ab’)2 fragment. PGN635 binds PS through the interaction of beta-2-glycoprotein 1 (β2GP1) in the same manner as bavituximab binding to β2GP1 in humans. Specific localization of NIR-labeled PS targeting antibodies after a single IV dose was observed in tumors compared to an isotype control antibody. Chemotherapy was shown to enhance the binding of PS targeting antibodies to tumors. These data provide a rationale to image PS expression to localize tumors or metastases and to monitor chemotherapy-induced PS expression during the course of bavituximab therapy.