Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC) are powerful methodologies to characterize the energetics of stability of DNA G-quadruplexes (G4s) and their interactions with potential drugs. G4s are nucleic acid secondary structures that form within guanine-rich sequences. Their formation in crucial points of genome has been associated with genetic diseases and cancer progression [1]. In addition, G4s are also important in the design of novel aptamers aimed at binding and inhibiting specific proteins [2]. G-quadruplexes are formed from G-rich sequences that build tetrads of hydrogen-bonded guanine bases, first found in telomeric DNA. In the last years, studies by Balasubramanian's group have showed that G-quadruplexes may exist in vivo; in addition, G-quadruplex formation in DNA is modulated during cell-cycle progression and endogenous G-quadruplex DNA structures can be stabilized by small-molecule ligands [3]. DSC can be utilized to study G4s of any molecularity providing all the thermodynamic parameters (enthalpy, entropy and free energy changes) for the folding/unfolding processes, without any two-step model assumption. In addition, comparison of the calorimetric enthalpies with calculated model-dependent enthalpies provides additional information on the unfolding process as the presence of intermediate states. DSC is important to determine the thermodynamic stability of the G4s and to evaluate the ability of the ligands to enhance their stability. Particularly, the Gibbs energy changes at physiological temperature in the presence and in the absence of ligands gives information on ligand stabilization upon binding process. On the other hand, ITC offers a direct measure of binding enthalpy, in addition to the stoichiometry and affinity constants. With the complete thermodynamic signature of drug-target interaction, dissecting the enthalpic and entropic components of binding is possible, which can be a useful aid to decision-making during drug optimization [4]. ITC can be used to rank all selected ligands in terms of their affinity towards the G-quadruplex targets.