The use of monoliths, one-piece porous separation materials, as column packaging media is known as monolithic chromatography and this technology has been widely exploited to improve the separation profile of high-valued biomolecules from complex biological matrices. Monoliths possess a wide range of processing advantages over commonly used chromatographical resins, including: fast flow rates, short processing times, sufficient surface area interactions, and enhanced thoroughness. The application of three different types of monolithic supports (hydrophobic, ion exchange and affinity) as an alternative to overcome traditional drawbacks in bioseparations will be presented as a practical perspective for the recovery of PEGylated proteins, stem cells or industrial enzymes. In our experience, the use of monolithic matrixes in the separation of high-valued biological materials has greatly simplified the usual problems observed in traditional chromatographic separations, resulting in a highly efficient downstream separation procedure. (C) 2016 Society of Chemical Industry