The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (H-bond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔHf). We report that introduction of a H-bond into the TC2═O group from the noncanonical nucleobase 2-aminopurine produces an expected decrease ∼10 ± 0.76 cm-1 (from ∼1720 cm-1 in Watson-Crick to ∼1710 cm-1 in 2-aminopurine), which correlates with an enthalpy of ∼0.93 ± 0.066 kcal/mol for this interaction.