The ability to isolate intact and functional mitochondria has greatly deepened our understanding of mitochondrial structure and function. With the advancement of molecular biology techniques and progression into omics-based research over recent decades, mitochondrial research has shifted from crop species such as wheat, pea, and potato to genetically sequenced models such as Arabidopsis thaliana and rice. Although there are many attributes that make model species particularly appealing for plant research, they are often less than ideal for conducting biochemical investigations and as such, considerable modification to mitochondrial isolation methods has been made. As the cost of genome sequencing continues to decrease however, an increasing number of crop species are now being sequenced and with these new resources it appears that the research community is turning back toward crop research. In this chapter we present mitochondrial isolation methods using density gradient centrifugation for both model species such as Arabidopsis thaliana, rice, and Medicago and crop species including wheat, potato, and pea. In addition, we present a number of marker enzyme assays to confirm mitochondrial purity as well as respiratory assays to determine outer membrane integrity and respiratory function of isolated mitochondria.