Electron beam (EB) curing-derived monolith materials were prepared from ethyl methacrylate (EMA), trimethylolpropane triacrylate (TMPTA), 2-propanol, 1-dodecanol, and toluene within the confines of 3 mm×100 mm id glass columns, applying a total dose of 22 kGy for curing. Monolithic columns were checked for their separation behavior for selected dansylated (DNS)-amino acids as well as for cyclophilin 18. Their separation performance was compared to that of a C18-modified silica-based rigid rod (Chromoliths®). In the separation of dansylated amino acids, retention times were reduced on EB-derived columns, where the peak resolution was significantly better than on a Chromolith®. This finding was attributed to a larger fraction of small pores (< 2.15 nm) in the EB curing-derived monoliths. Finally, EB curing-derived monoliths have been used to separate cyclophilin 18 from crude cell lysis mixtures.