Mitochondrial respirometry is key to understanding how environmental factors model energetic cellular processes. Until now, no reports have shown temperature effects and other environmental variables on cephalopod mitochondria activity because of the lack of a method to evaluate mitochondrial respiratory parameters on those groups of species. In this sense and for the first time, it showed the mitochondrial respirometry data of adult Octopus maya’s heart. Following the protocol is illustrated a step-by-step procedure to get the corresponding respiratory parameters.