In this study, we demonstrated that microwave-assisted enzymatic digestion could be greatly accelerated by multifunctional magnetite beads. The acceleration of microwave-assisted enzymatic digestion by the presence of the magnetite beads was attributable to several features of the beads. Their capacity to absorb microwave radiation leads to rapid heating of the beads. Furthermore, their negatively charged functionalities cause adsorption of proteins with opposite charges onto their surfaces by electrostatic interactions, leading to a concentration on the surfaces of the beads of proteins present in trace amounts in the solution. The adsorbed proteins are denatured and hence rendered vulnerable to enzymatic digestion and are digested on the beads. For microwave heating, 30 s was sufficient for carrying out the tryptic digestion of cytochrome c, in the presence of magnetite beads, while 1 min was adequate for tryptic digestion of myoglobin. The digestion products were characterized by MALDI-MS. This rapid enzymatic digestion allowed the entire time for identification of proteins to be greatly reduced. Furthermore, specific proteins present in trace quantities were enriched from the sample on the magnetite beads and could be rapidly isolated from the sample by employing an external magnetic field. These multiple roles of magnetite beads, as the absorber for microwave irradiation, the concentrating probe, and the agent for unfolding proteins, contributed to their capability of accelerating microwave-assisted enzymatic digestion. We also demonstrated that trypsin immobilized magnetite beads were suitable for use in microwave-assisted enzymatic digestion.