Monoliths media are gaining interest as excellent substitutes to conventional particle-packed columns. Monolithic columns show higher permeability and lower flow resistance than conventional liquid chromatography columns, providing high-throughput performance, resolution and separation in short run times. Monolithic columns with longer length, smaller inner diameter and specific selectivity to peptides or enantiomers have been played important role in hyphenated system. Monolithic stationary phases possess great efficiency, resolution, selectivity and sensitivity in the separation of complex biological samples, such as the complex mixtures of peptides for proteome analysis. The development of monolithic stationary phases has opened the new avenue in chromatographic separation science and is in turn playing much more important roles in the wide application area. Monolithic stationary phases have been widely used in fast and high efficiency one- and multi-dimensional separation systems, miniaturized devices, and hyphenated system coupled with mass spectrometers. The developing technology for preparation of monolithic stationary phases is revolutionizing the column technology for the separation of complex biological samples. These techniques using porous monoliths offer several advantages, including miniaturization and on-line coupling with analytical instruments. Additionally, monoliths are ideal support media for imprinting template-specific sites, resulting in the so-called molecularly-imprinted monoliths, with ultra-high selectivity. In this review, the origin of the concept, the differences between their characteristics and those of traditional packings, their advantages and drawbacks, theory of separations, the methods for the monoliths preparation of different forms, nanoparticle monoliths and metal-organic framework are discussed. Two application areas of monolithic metal-organic framework and nanoparticle monoliths are provided. The review article discusses the results reported in a total of 218 references. Other older references were included to illustrate the historical development of monoliths, both in preparation and types, as well as separation mechanism.