In several cases, the high water content in the mobile phase, required for the retention of polar analytes, decreases the sensitivity in API-MS because of the inappropriate ionization conditions (2). Alternatively, polar endcapped reversed-phase stationary phases are commonly used stationary phases with embedded polar groups (the so-called "aqueous" phases), which are stable in high water content, are also used. Despite this, such investigations with low-organic-content eluent may cause several problems because of the risk of folding for the hydrophobic alkyl chains and the expulsion of eluent from the pore spaces. Appropriate elution conditions with high water content or ion-pair reagents have been developed for the retention of polar analytes. Polar and hydrophilic compounds are hard to analyze by classical reversed-phase stationary phases and they are usually eluted near the hold up volume of the column. Reversed-phase HPLC is among the most popular liquid chromatographic techniques, in which the separation is mainly based on hydrophobic interactions between the solutes and the hydrophobic stationary phase thus, apolar or weakly polar compounds are preferentially retained. Here, we discuss the principles of HILIC and reversed-phase HPLC, the recent advancements of HILIC×reversed-phase HPLC–API-MS (orthogonal) and HILIC–reversed-phase HPLC–API-MS (linear) hyphenation for the analysis of complex mixtures and its future perspectives. Highly accurate mass spectrometers can be used to obtain empirical formulas for molecules up to 1000 Da and using tandem MS further leads to structural information by molecular fragmentation. Because of the complexity of the mixtures, integration of HPLC systems with atmospheric-pressure-ionization (API) mass spectrometry (MS), used as an identification method based on the molecular weight of the analytes, seems to be preferred. Recent studies have presented interesting on-line coupling of reversed-phase HPLC with HILIC for the analysis of both apolar and polar compounds, such as proteins, drugs, and metabolites in natural samples. Its increasing popularity is mainly because of the complementary selectively to the well-known reversed-phase HPLC, which is extensively used for the analysis of hydrophobic analytes. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new technique for the analysis of polar compounds. In this regard, the orthogonal selectivity of the stationary phases is one of the most important parameters for an efficacious hyphenation. The hyphenation of two or more columns with different retention mechanisms in multidimensional chromatography is an efficient approach, and was initially developed for proteome analysis (1). In general, one-dimensional chromatography, in which the separation is achieved with a single column, is not enough to resolve complex mixtures. In this context, high performance liquid chromatography (HPLC), one of the most powerful separation techniques based on the different physicochemical properties of the compounds, is being widely used. These studies have prompted, and are still prompting, the development of new analytical methods and settings to investigate compounds in a wide range of polarity. The so-called "entire component" analysis is particularly required in growing research fields such as metabolomic or environmental analysis that follow a nontargeted approach, in which no hypothesis is formulated about the compound to investigate. In recent years, there has been an increased necessity to collect information in a single analysis about a huge number of compounds present in complex mixtures. Thus, a hyphenation of both liquid chromatographic techniques with atmospheric pressure ionization (API) and mass spectrometry (MS) seems to be a consequent step. Both separation variants can be operated easily with volatile and water-miscible solvents (for example, ammonium acetate aqueous solution and acetonitrile). Hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high performance liquid chromatography (HPLC) are complementary techniques in the separation of organic molecules with a broad band of polarity.