Mass spectrometry (MS) is a powerful analytical technique that is now widely used in the chemical, physical, engineering, and life sciences, with rapidly growing applications in many areas including clinical, forensic, pharmaceutical, and environmental fields. The increase in use of MS in both academic and industrial settings for research and development over the past decade has created excellent employment opportunities for those conversant with the technique. Electrospray ionization mass spectrometry (ESI-MS) is applicable to a wide range of molecules, from drugs to proteins, and is easy to use. ESI-MS can be used in either the positive or negative mode to create gas-phase ions from positively and negatively charged molecules; however, there is a lack of laboratory experiments that introduce students to negative ion ESI-MS and tandem mass spectrometry (MS/MS). This experiment investigates the complexation between NADH and boric acid using negative ion ESI-MS. The experiment then uses MS/MS to determine the site of boric acid binding to the nucleotide, demonstrating the power of gas-phase fragmentation patterns for structural assignments. (Contains 1 table and 4 figures.)