F1F0 ATP synthase, complex V of the oxidative phosphorylation pathway, is the enzyme that phosphorylates ADP into ATP using the electrochemical energy of the proton gradient generated by the respiratory chain. It produces the vast majority of cellular ATP. We describe an improved spectrophotometric assay for its activity, measured in its reverse reaction i.e. ATP hydrolysis. Using this procol in four different murine organs: brain, heart, muscle and liver, we obtained organ-dependent, reproducible and stable complex V specific activities, similar with fresh and frozen organs. Similar inhibition by oligomycin and exogenous IF1 demonstrated tight coupling between F1 and F0 domains. The activity was stable for several hours. It was linear with protein amount. A schematic representation of the assay is provided below.