Testing the predictions of the Mitochondrial Free Radical Theory of Ageing (MFRTA) has provided a deep understanding of the role of reactive oxygen species (ROS) and mitochondria in the aging process. However those data, which support MFRTA are in the majority correlative (e.g. increasing oxidative damage with age). In contrast the majority of direct experimental data contradict MFRTA (e.g. changes in ROS levels do not alter longevity as expected). Unfortunately, in the past, ROS measurements have mainly been performed using isolated mitochondria, a method which is prone to experimental artifacts and does not reflect the complexity of the in vivo process. New technology to study different ROS (e.g. superoxide or hydrogen peroxide) in vivo is now available; these new methods combined with state-of-the-art genetic engineering technology will allow a deeper interrogation of, where, when and how free radicals affect aging and pathological processes. In fact data that combine these new approaches, indicate that boosting mitochondrial ROS in lower animals is a way to extend both healthy and maximum lifespan. In this review, I discuss the latest literature focused on the role of mitochondrial ROS in aging, and how these new discoveries are helping to better understand the role of mitochondria in health and disease. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.