ATP synthase is crucial for ATP synthesis in living cells. Recently, ATP synthase was found not only in mitochondria but also on the extracellular surface, named as ectopic ATP synthase (ecto-ATP synthase). ATP synthase inhibitor is a potential drug candidate to fight cancer by blocking the ecto-ATP synthase of cells. In this study, we applied dynamic phosphoproteomics to elucidate the molecular responses to ecto-ATP synthase blockade.