G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG₃(T₂AG₃)₃]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy ($\mathrm{\Delta }{G}_{\mathrm{b}}^0$ = −10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands. Significance A thorough characterization of the binding interaction between a drug and its molecular target is fundamental to successfully lead drug design. We demonstrate that this characterization is also possible using the recently developed method of funnel-metadynamics (FM), here applied to investigate the binding of berberine to DNA G-quadruplex. We computed a quantitatively well-characterized free-energy landscape that allows identifying two low-energy ligand binding modes and the presence of higher energy prebinding states. We validated the accuracy of our calculations by steady-state fluorescence experiments. The good agreement between the theoretical and experimental binding free-energy value demonstrates that FM is a most reliable method to study ligand/DNA interaction.