Since its discovery, electrospray ionization mass spectrometry (ESI-MS) has provided valuable information on structurally specific biomolecular interactions, including DNA-drug, enzyme-inhibitor, DNAprotein and protein-protein interactions. In addition, this technique has emerged as a powerful tool in probing the structural modification of large molecules. However, ESI-MS has been underutilized in analysis and mechanistic studies involving small molecules. Here, a brief overview is presented to familiarize non-specialists with ESI-MS applications in analysis and mechanistic studies of small molecules. For more advanced applications (e.g. biopolymers) or detailed analytical descriptions of the ionization processes and the equipment, readers are referred to a number of excellent reviews and books.