AbstractThe advent of soft ionization techniques, notably electrospray and laser desorption ionization methods, has enabled the extension of mass spectrometric methods to large molecules and molecular complexes. This both greatly extends the applications of mass spectrometry and makes the activation and dissociation of complex ions an integral part of these applications. This review emphasizes the most promising methods for activation and dissociation of complex ions and presents this discussion in the context of general knowledge of reaction kinetics and dynamics largely established for small ions. We then introduce the characteristic differences associated with the higher number of internal degrees of freedom and high density of states associated with molecular complexity. This is reflected primarily in the kinetics of unimolecular dissociation of complex ions, particularly their slow decay and the higher energy content required to induce decomposition—the kinetic shift (KS). The longer trapping time of Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) significantly reduces the KS, which presents several advantages over other methods for the investigation of dissociation of complex molecules. After discussing general principles of reaction dynamics related to collisional activation of ions, we describe conventional ways to achieve single‐ and multiple‐collision activation in FT‐ICR MS. Sustained off‐resonance irradiation (SORI)—the simplest and most robust means of introducing the multiple collision activation process—is discussed in greatest detail. Details of implementation of this technique, required control of experimental parameters, limitations, and examples of very successful application of SORI‐CID are described. The advantages of high mass resolving power and the ability to carry out several stages of mass selection and activation intrinsic to FT‐ICR MS are demonstrated in several examples. Photodissociation of ions from small molecules can be effected using IR or UV/vis lasers and generally requires tuning lasers to specific wavelengths and/or utilizing high flux, multiphoton excitation to match energy levels in the ion. Photodissociation of complex ions is much easier to accomplish from the basic physics perspective. The quasi‐continuum of vibrational states at room temperature makes it very easy to pump relatively large amounts of energy into complex ions and infrared multiphoton dissociation (IRMPD) is a powerful technique for characterizing large ions, particularly biologically relevant molecules. Since both SORI‐CID and IRMPD are slow activation methods they have many common characteristics. They are also distinctly different because SORI‐CID is intrinsically selective (only ions that have a cyclotron frequency close to the frequency of the excitation field are excited), whereas IRMPD is not (all ions that reside on the optical path of the laser are excited). There are advantages and disadvantages to each technique and in many applications they complement each other. In contrast with these slow activation methods, the less widely appreciated activation method of surface induced dissociation (SID) appears to offer unique advantages because excitation in SID occurs on a sub‐picosecond time scale, instantaneously relative to the observation time of any mass spectrometer. Internal energy deposition is quite efficient and readily adjusted by altering the kinetic energy of the impacting ion. The shattering transition—instantaneous decomposition of the ion on the surface—observed at high collision energies enables access to dissociation channels that are not accessible using SORI‐CID or IRMPD. Finally, we discuss some approaches for tailoring the surface to achieve particular aims in SID. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:135–167, 2005