Hydrophilic interaction liquid chromatography (HILIC) has recently become more important, particularly for the analysis of polar drugs, metabolites and biologically relevant compounds in glycomics, proteomics, metabolomics and clinical analysis. HILIC makes it possible to increase the retention of polar coumpounds, achieve orthogonal selectivity and increase mass spectrometry (MS) sensitivity, compared with reversed-phase liquid chromatography. This article discusses the advantges and limitations of HILIC in a variety of practical applications in the pharmaceutical industry.