Abstract <p>Aptamer-based pH switches hold significant potential for targeting cancer cells, which exhibit distinct pH microenvironments. Studying the operation mechanisms of such switches is a prerequisite for their rational design. However, the thermodynamics of aptamer-based pH switches remains challenging to explore due to aptamer target instability, laboriousness, uncertainty, or unavailability. This work introduces a novel approach that uses short nucleic acid mimetics to probe the pH-dependent behavior of aptamer switches without requiring the target molecule. By mimicking ATP binding, these probes enable the analysis of thermodynamic transitions in a model ATP aptamer-based pH-dependent system. Two complementary approaches—free energy scanning and melting curve fitting—were developed to determine effective dissociation constants and the corresponding Gibbs free energy changes for ATP binding to a set of pH switches. The results showed that the proposed techniques are applicable to reliably determine the Gibbs free energy changes of specified switch states with high precision. The mimetic-based strategy circumvented the limitations of conventional aptamer target application and demonstrated consistency with prior studies. This method provides a versatile framework for the rational analysis of environment-responsive molecular devices, advancing applications in diagnostics and targeted therapies.</p> Graphical abstract <p>The principle of mimetics application</p>