The placenta consists of multiple cell layers, including the underlying cytotrophoblast layer and a multi-nucleated, terminally differentiated syncytiotrophoblast. These two cell types possess mitochondria with unique properties; differing in size, morphology, energy production and steroidogenic capacity [[1], [2], [3]]. Mitochondria are vital to virtually every aspect of cellular function from supplying energy in the form of ATP, processing metabolic intermediates, modulating Ca2+ signalling, controlling ROS production and playing a critical anabolic role. Many studies have shown that mitochondrial dysfunction contributes to placental pathology underpinning gestational disorders [4,5]. However, due to the complex cell structure of the placenta and the very different biology of cytotrophoblast and syncytiotrophoblast, mitochondrial populations from both are often studied together so subtle variations are missed. One of the key challenges to date has been the development of methodologies for isolation of functional mitochondria that allow key parameters to be assessed in an efficient and reproducible way [3]. Therefore, the aim of this study was to develop methodologies for the isolation, cryopreservation and biochemical analysis of mitochondria from both cytotrophoblast and the syncytiotrophoblast.