Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.