Long non-coding RNAs (lncRNAs) play a key role in numerous biological processes, including gene regulation. One potential mechanism involves the formation of RNA•DNA:DNA triplex structures, where the lncRNA binds in the major groove of a target DNA via Hoogsteen base pairs. Here, we investigated the impact of the lncRNA binding on the stability of the DNA duplex target to gain more insights into the triplex formation process. Quantification of the temperature-dependent imino proton solvent exchange allows the determination of the changes of individual DNA duplex base pairs upon triplex formation. The data shown here demonstrate that triplex formation alters DNA structure and stability by affecting both hydrogen bonding and base-stacking interactions. These thermodynamic insights support the prediction of triplex stability and enhance the understanding of RNA•DNA:DNA triplex formation.