Introduction 1 2 7 8 9 10 11 13 14 12 15 Although HILIC applications cover a large range of bioactive compounds, in this review we will focus on the use of HILIC in proteomics. We will describe how HILIC can be used for the separation of peptides in multidimensional chromatography and provide insights into its sensitivity, selectivity, separation power and “orthogonality.” We will also discuss how HILIC has recently been used for the enrichment of post-translational modifications at both peptide and protein levels. HILIC 16 1 2 9 17 18 2 9 19 20 17 18 21 22 23 24 25 14 26 1 10 Fig. 1 Chemical structures of the functional groups in common HILIC stationary phases  17 2 17 2 10 2 10 4 2 23 17 10 10 4 23 4 23 23 23 23 27 K a 10 12 12 12 2 HILIC in multidimensional peptide separation 28 29 30 31 34 35 36 37 36 11 12 38 39 38 40 40 11 2 11 Fig. 2 11  12 11 2 11 12 11 12 12 12 41 41 45 42 9 11 HILIC for the analysis of PTMs 46 14 47 48 49 50 52 47 HILIC in the targeted analysis of phosphorylated peptides 53 47 54 55 50 54 2 51 52 56 57 58 15 22 23 27 15 22 23 22 12 2 HILIC in the targeted analysis of N-terminally acetylated peptides 59 60 33 54 61 12 3 12 12 Fig. 3 bare line squares triangles N 12  12 33 12 HILIC in the targeted analysis of glycosylated peptides 62 63 64 65 14 66 66 14 67 68 14 66 69 72 4 70 Fig. 4 upper spectrum lower spectrum 70  69 66 14 18 66 69 14 66 C. elegans 72 72 66 65 70 73 64 74 HILIC in the targeted analysis of histone modifications 75 76 77 5 7 21 78 80 5 6 79 81 77 82 82 77 5 Fig. 5 77  In these histone analyses, HILIC delivered the extra separation step that was needed to sufficiently reduce the complexity of the samples and to separate the most important histone modifications. Concluding remarks Recent years have witnessed an increased interest in HILIC. More versatile and diverse stationary phases have become available, leading to reports of an exciting and broad range of applications. The unique separation and orthogonality of HILIC towards RP (the most commonly used peptide separation method) make it an ideal method for multidimensional chromatography that can extend separation power. As far as selectivity is concerned, HILIC can compete well with RP and SCX, which are the two main chromatographic techniques applied today. Focusing solely on proteomics applications, HILIC has been shown to be very versatile in analyses of protein modifications. The retention of hydrophilic compounds can be effectively exploited in the enrichment of phosphorylated, N-terminally blocked and glycosylated peptides. Moreover, in combination with RP it allows the separation of complex differentially modified histones, an incredibly challenging area. It is therefore expected that the number of applications of HILIC in the proteomics field will increase significantly in the years to come, and that the development of novel HILIC stationary phases and/or monolithic columns will continue. Much progress is expected.