<p>Isothermal titration calorimetry (ITC) is the gold standard for thermodynamic characterization of molecular interactions, and more recently has allowed for the provision of kinetic information in a single in-solution and label-free measurement. While extensively applied to protein-protein and protein-ligand interactions, ITC remains underutilized in nucleic acid research, despite its ability to resolve complex molecular binding events. In this study, we leverage ITC to characterize the formation of DNA triplexes, a class of non-canonical DNA structures involved in gene regulation and genomic integrity. Crucially, this study represents the first quantitative determination of kinetic parameters for triplex DNA formation using ITC, offering unprecedented insights into the dynamic aspects of these interactions. By integrating ITC data with global fitting models, we extract both thermodynamic and kinetic parameters, revealing how sequence composition, strand length, and pH influence triplex stability. The exceptional sensitivity of ITC allows us to detect subtle energetic differences that conventional spectroscopic techniques often overlook, establishing ITC as a superior tool for nucleic acid research. Our findings not only validate ITC as an indispensable method for studying triplex DNA but also open new avenues for investigating nucleic acid assembly kinetics with high precision.</p>