Purpose The objective is to develop a real-time Mitochondrial ATP contents and Response Against Inhibiting and Stimulating Substrates (MitoRAISE) assay and assess its potential to evaluate the mitochondrial oxidative phosphorylation function. Methods MitoRAISE measures the total ATP contents, ATP synthesis capacity, and the response to inhibitory substrates. The measurements were all quantified with an ATP standard curve and validated with in vitro testing. MitoRAISE was then applied using peripheral blood mononuclear cells (PBMCs) from 35 healthy volunteers and 20 breast cancer patients. Result Results from isolated mitochondria, cells with different permeabilization percentages, and cells with damaged mitochondria demonstrated the ability of capturing ATP signals specifically from the mitochondria. Breast cancer PBMCs exhibited a significant increase in glutamic acid- and malic acid-induced mitochondrial ATP synthesis capacity yet a significant decrease in mitochondrial DNA copy number (mtDNA CN) compared to healthy PBMCs. Furthermore, breast cancer PBMCs mostly showed negative correlation between mtDNA CN and parameters from MitoRAISE but healthy individuals showed positive correlation. Conclusion We developed a quick and easy method to detect real-time mitochondrial activity in live cells. Monitoring mitochondrial ATP synthesis capacity and sensitivity to inhibitory substrates could aid in assessing the functional status of mitochondrial oxidative phosphorylation.