Description: Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and 99m Tc- and 111 In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%–5%). There was a significant correlation ( R 〉 0.9, P 〈 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99m Tc-C2Am and 111 In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99m Tc-C2Am and 111 In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3 x and 2.2 x , respectively, at 2 h and 7.3 x and 4.1 x , respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99m Tc- and 111 In-labeled C2Am, and their ability to produce rapid and cell death–specific image contrast, these agents have potential for clinical translation.