All cells rely on ATP to perform their basic functions. However, the maintenance of active motility imposes particularly high ATP demands on spermatozoa. Energy demands vary during the life of the sperm cell and they are crucial when spermatozoa travel in the female tract and undergo processes essential for fertilization, such as capacitation, hyperactivation, and acrosomal exocytosis. Spermatozoa produce ATP via glycolysis and/or oxidative phosphorylation (OXPHOS) and each of these pathways are localized to different regions of the sperm flagellum. Species vary in their use of these two pathways for ATP production, and these can change during the different life stages. Here, we present methods to characterize bioenergetics of spermatozoa. A protocol is given to quantify ATP levels by using a bioluminescence assay kit in extracts from sperm cells incubated under non-capacitating or capacitating conditions. An assay to measure lactate excreted to the incubation medium, using colorimetric/fluorometric kit, is given to infer the potential contribution of the glycolytic pathway. Lastly, we present the protocol to analyze the energy metabolism of live sperm, which can be used to determine the relative contributions of glycolysis and OXPHOS, based on extracellular flux analysis.