Adenosine triphosphate is a major vector of chemical energy in living organisms, but its detection is sometimes hindered by complicated physiological sample matrixes. In this work, we demonstrated a ratiometric bioassay for the accurate and sensitive detection of ATP by measuring the 197Au/115In signal ratio of a mass spectrometric core-satellite structure. Validation of the proposed bioassay was successfully demonstrated in cell lysates and human serum samples.