In recent years, monolithic columns have been widely used as efficient stationary phases due to some advantages over particle-based counterparts such as their relative ease of preparation and good permeability. Among other types of monoliths, the use of polymethacrylate-based monoliths in separation science has gained popularity in the last two decades due to the fact that they are easy to fabricate and do not require retaining frits. The polymethacrylate-based monoliths have also been investigated with various chromatographic modes. These type of monoliths present high potential as stationary phases for the separation and purification of biomolecules such as amino acids, peptides, proteins, and nucleic acids. This review summarizes the biochromatographic applications of polymethacrylate-based monoliths in the separation and analysis of small and large biomolecules in both the various HPLC formats from nano-HPLC to large-scale HPLC and capillary electrochromatography. This review is not fully comprehensive but is intended to provide an overview of the different aspects of polymethacrylate monolith applications.