Background: The inhibition of autophagy using pharmacological inhibitors such as chloroquine may be an effective strategy to overcome chemotherapy or resistance to anti-angiogenic therapy. Materials and Methods: The cytotoxic effect of doxorubicin (0.1-1 mu M), chloroquine (0.25-32 ,mu M) and their combination were investigated by employing ATP assay in human umbilical vein endothelial cells (HUVECs). The effect of doxorubicin and chloroquine combination was also measured using tube formation assay on Matrigel. The anti-angiogenic activities of doxorubicin (2.5 mu g/pellet) and chloroquine (15 mu g/pellet), their combination, and standards (50 mu g/pellet) were tested in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Results: The combination of doxorubicin and chloroquine significantly had a stronger anti-angiogenic effect than the positive control (+/-)-thalidomide and doxorubicin alone in the CAM assay and in vitro tube-formation assay. Conclusion: Chloroquine enhanced the anti-angiogenic effect of doxorubicin on CAM at the tested concentrations.