Mineralizing matrix vesicles (MVs) are extracellular organelles produced by chondrocytes, osteoblasts, and odontoblasts under physiological conditions and by vascular smooth muscle cells under pathological conditions. MVs are involved in the early stage of mineralization allowing calcium and phosphate to accumulate, and therefore providing an optimal environment facilitating hydroxyapatite formation. Here, we describe the isolation of MVs from osteoblasts and chondrocytes and present their main characteristics.