© 2015, World Molecular Imaging Society. Purpose: The aim of the study was to develop a reliable quantitative imaging biomarker from fluorescence microscopic imaging of TRA-8/death receptor 5 (DR5) oligomer to predict TRA-8 therapeutic efficacy in human breast and pancreatic cancer mouse models. Procedures: Two breast (2LMP, SUM159) and two pancreatic (MIA PaCa-2, PANC1) cancer cell lines were used. 105 cells per cell line were placed in a culture dish and treated with Cy5.5-labeled TRA-8 overnight in vitro. Three fluorescence microphotographs (×20) were acquired from randomly selected areas, and about 300 cells were analyzed per cell line. Two-dimensional (2D) fluorescence signal distribution of Cy5.5-TRA-8 on each cell was measured. Gaussian curve fitting to the distribution was determined by the least square regression method, and the coefficient of determination (R2) of the fitting was found. In addition, two features of the best fitting Gaussian curve such as peak amplitude and the volume under the curve (VUC) were retrieved. A novel image biomarker was extracted by correlating the combination of R2 value, peak amplitude, and the VUC with the logarithmic values of the half maximal inhibitory concentrations (IC50) of TRA-8 for the four cell lines or the percentage of tumor growth inhibition (%TGI) at a week of TRA-8 treatment in animal models. Results: Cy5.5-TRA-8 binding to DR5 receptors resulted in an oligomer on each cell membrane, and its fluorescence signal distribution followed Gaussian curve. Peak amplitude of fluorescence signal in the oligomeric region, R2 value of the Gaussian fitting, and the VUC in TRA-8-sensitive cells were significantly higher than those in resistant cells (p < 0.05). The novel imaging biomarker was significantly correlated with either log10(IC50) or %TGI (p < 0.001). Conclusion: The imaging biomarker extracted from the cellular distribution pattern of Cy5.5-TRA-8 may serve as a predictive biomarker of TRA-8 therapy for cancer patients.