The investigation of vascular calcification and its underlying cellular and molecular pathways is of great interest in current research efforts. Therefore, suitable assays are needed to allow examination of the complex calcification process under controlled conditions. The current study describes a new ex vivo model of isolated-perfused rat aortic tissue with subsequent quantification and vessel staining to analyze the calcium content of the aortic wall. A rat aorta was perfused ex vivo with control and calcification media for 14 days, respectively. The calcification medium was luminally perfused and induced a significant increase in calcium deposition within the media of the vessel wall detected alongside the elastic laminae. Perfusion with control medium induced no calcification. In addition, the mRNA expression of the osteogenic marker bone morphogenetic protein 2 (BMP-2) increased in aortic tissue after perfusion, while SM22α as smooth muscle marker decreased. This newly developed ex vivo model of isolated-perfused rat aorta is suitable for vascular calcification studies testing inducers and inhibitors of vessel calcification and studying signaling pathways within calcification progression.