Mitochondrial content and basal respiration have been repeatedly demonstrated to be higher in leukemia. Although these findings are suggested to reflect higher reliance on oxidative ATP production, bioenergetic efficiency in leukemic mitochondria has not been empirically tested. That is, it remains unclear if higher basal respiration in leukemia reflects accelerated demand for ATP regeneration andor intrinsic limitations in bioenergetic efficiency. Distinguishing between these potential outcomes is critical, as the molecular cause and thus potential treatment strategies are distinct. Herein, we sought to comprehensively evaluate bioenergetic efficiency in blood cancer mitochondria using a discovery-based biochemical platform. This platform encompasses two key technological advancements 1 comprehensive unbiased analysis similar in scope to traditional omics, and 2 utilization of the creatine kinase clamp that allows assays to be performed across physiological ATP free energies deltaGATP, thus permitting quantitative evaluation of bioenergetic efficiency. Experiments were performed in intact cells, permeabilized cells, and isolated mitochondria derived from human leukemia HL-60, KG-1, and MV-4-11, as well as peripheral blood mononuclear cells PMBCs. Results revealed three key insights related to substrate preference, electron transport regulation, and bioenergetic efficiency.