This review summarizes the preparation and application of chromatographic separation media based on methacrylate monomers with a major focus on highly crosslinked macroporous beads prepared from 2-hydroxyethyl methacrylate and glycidyl methacrylate, respectively. The effects of process variables such as composition of the polymerization mixture that includes monomers, porogenic solvents, and free radical initiator, suspension stabilizer, reaction temperature, and stirring are detailed for both classical and templated suspension polymerization. In addition, specific features of the preparation of monodisperse beads are also discussed. The performance of methacrylate-based separation media is demonstrated on numerous separations in a variety of chromatographic modes.