Chromatography is one of the methods for separation and purification of biomolecules, and chromatography media has been widely concerned as a key part of the chromatography process. The pore structure of the chromatographic medium has a significant influence on its separation and purification efficiency. Therefore, it is very important to explore the pore structure of the chromatographic medium. This paper introduced several methods commonly used to determine the pore diameter structure of chromatographic media, such as mercury intrusion porosimetry, nitrogen adsorption, small angle X-ray scattering, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and inverse size-exclusion chromatography (ISEC). The basic principle and common probe types of ISEC are described, the advantages and disadvantages in the application of ISEC are summarized, the development history of ISEC and its research advance are introduced in this paper.