Hydrophilic interaction liquid chromatography (HILIC) is a powerful separation technique for polar compounds on polar stationary phases. This review discusses the characterization of HILIC stationary phases and their applications in separating polar compounds in complex matrices. HILIC is an alternative to normal-phase liquid chromatography (NP-LC) and reversed-phase liquid chromatography (RP-LC), offering advantages such as better sensitivity and the ability to analyze charged substances. HILIC uses traditional polar stationary phases like silica, amino, or cyano, and mobile phases similar to those in RP-LC. It can be used in orthogonal or two-dimensional separations and has applications in bioanalysis. HILIC is particularly effective for separating highly hydrophilic and amphiphilic compounds that are difficult to retain in RP-LC. The separation mechanism in HILIC involves hydrophilic partitioning and can also include ion exchange and hydrogen bonding. HILIC has gained popularity due to its versatility and compatibility with mass spectrometry. The review highlights the importance of understanding retention behavior in HILIC to expand its applications. HILIC is used for the analysis of carbohydrates, peptides, and polar pharmaceuticals. The review also discusses the characterization of HILIC stationary phases and their selectivity for polar compounds. The separation mechanism in HILIC is influenced by the physicochemical properties of the stationary phase and mobile phase, as well as the sample structure. HILIC is an ideal method for multidimensional chromatography due to its orthogonality with RP-LC. It is particularly suited for solutes without charge and has applications in pharmaceuticals, proteomics, metabolomics, and medical science. HILIC is widely used in bioanalytical applications due to its compatibility with mass spectrometry and its ability to separate polar compounds. The review concludes that HILIC is a versatile and effective technique for separating polar compounds and has a broad range of applications in various fields.Hydrophilic interaction liquid chromatography (HILIC) is a powerful separation technique for polar compounds on polar stationary phases. This review discusses the characterization of HILIC stationary phases and their applications in separating polar compounds in complex matrices. HILIC is an alternative to normal-phase liquid chromatography (NP-LC) and reversed-phase liquid chromatography (RP-LC), offering advantages such as better sensitivity and the ability to analyze charged substances. HILIC uses traditional polar stationary phases like silica, amino, or cyano, and mobile phases similar to those in RP-LC. It can be used in orthogonal or two-dimensional separations and has applications in bioanalysis. HILIC is particularly effective for separating highly hydrophilic and amphiphilic compounds that are difficult to retain in RP-LC. The separation mechanism in HILIC involves hydrophilic partitioning and can also include ion exchange and hydrogen bonding. HILIC has gained popularity due to its versatility and compatibility with mass spectrometry. The review highlights the importance of understanding retention behavior in HILIC to expand its applications. HILIC is used for the analysis of carbohydrates, peptides, and polar pharmaceuticals. The review also discusses the characterization of HILIC stationary phases and their selectivity for polar compounds. The separation mechanism in HILIC is influenced by the physicochemical properties of the stationary phase and mobile phase, as well as the sample structure. HILIC is an ideal method for multidimensional chromatography due to its orthogonality with RP-LC. It is particularly suited for solutes without charge and has applications in pharmaceuticals, proteomics, metabolomics, and medical science. HILIC is widely used in bioanalytical applications due to its compatibility with mass spectrometry and its ability to separate polar compounds. The review concludes that HILIC is a versatile and effective technique for separating polar compounds and has a broad range of applications in various fields.