CD73 is an ectonucleotidase able to catabolize 5'-adenosine monophosphate (AMP) into adenosine at the extracellular level. Extracellular adenosine plays a critical role in regulating many processes under physiological and pathological conditions. In the context of cancer, the expression and activity of CD73, either in tissue and in biological fluids, is increased leading to high levels of adenosine that potently suppress T-cell mediated responses, promoting tumor progression through stimulation of adenosine receptors. Compelling evidence indicates that elevated levels of CD73-generating adenosine limit the efficacy of cancer immunotherapy. Inhibitors of ectonucleotidases and antagonists of adenosine receptors have emerged as new therapeutic tools to improve anti-tumor immune response and potentially synergize with currently used immunotherapeutic agents. Measurement of CD73 levels in serum of cancer patients is a promising approach that, although it needs to be validated, may help to select patients who will benefit from adenosine-targeting agents and predict response to immunotherapy. Here, we describe a simple and fast method to evaluate the AMPase activity of CD73 in peripheral blood that may also be applied to other biological fluids.