Mitochondria are the powerhouse of animal cells. They produce through oxidative phosphorylation more than 90% of the cellular energy (ATP) required for organism's growth, reproduction, and maintenance. Hence, information on mitochondrial function is expected to bring important insights in animal ecology and evolution. Unfortunately, the invasiveness of the procedures required to measure mitochondrial function (e.g. sampling of liver or muscles) has limited its study in wild vertebrate populations so far. Here, we capitalize on the fact that bird red blood cells (RBCs) possess functional mitochondria to describe a minimally invasive approach to study mitochondrial function, using blood samples. In the king penguin, we present a protocol, using a high-resolution respirometry system and specific agonists and antagonists enabling the assessment of mitochondrial function in RBCs. We evaluated the inter-assay repeatability of our measures of mitochondrial function, and tested the influence of sample storage and bird handling time on these measures. We also compared measures of mitochondrial function in RBCs and in the pectoral muscle obtained from the same individuals. Mitochondria from RBCs showed the expected responses to mitochondrial agonists and antagonists, and therefore the protocol presented allows computing effective measures of mitochondrial function. The different measures of RBC mitochondrial function were significantly repeatable, were not affected by the handling time of the bird prior to blood sampling (i.e. stress response), and only minimally affected by the storage time of the sample at 4 °C up to 24 h. Most notably, we showed that mitochondrial parameters measured in RBCs moderately correlated to those measured in the pectoral muscle. This study sheds light on the use of RBCs in birds as a valuable and minimally invasive source of information on mitochondrial function. This approach opens new opportunities to study mitochondrial function in free-living animals and could bring knowledge gains in ecology and evolution. Fish, amphibians and reptiles also possess mitochondria in their RBCs, and the approach presented here could also be applicable to these taxa.