Understanding the characteristics, role and structure of nucleic acids allowed to answer questions about the disease processes. Today, nucleic acids and their constituents are tools, which are used by molecular biology in medicine and biotechnology. Antisense and gene therapy are intensively developing methods for possible treating or preventing disease. They use short fragments of DNA or RNA - oligonucleotides to silence the genes expression. They are not the only ones that allow analytical chemists to obtain information about the state of our body. Determination of modified nucleoside allows detection of cancer, while analysis of nucleotides allows the estimation of strengthening the immune system. There is a great need of sensitive, selective and precise methods of separation of nucleosides, nucleotides and oligonucleotides and their qualitative and quantitative analysis. Consequently liquid chromatography (LC) is the most commonly used for analysis of nucleic acid constituents. The most widely used modes of LC include Ion Exchange Chromatography (IEC) and Reversed Phase High Performance Liquid Chromatography (RP HPLC). Both techniques have their advantages and disadvantages in the analysis of nucleosides, nucleotides and oligonucleotides. In the case of IEC it is necessary to use high concentrations of the salt in the mobile phase or concentration gradients, which considerably limits the possibility of using MS detection. RP HPLC can be coupled with MS detection but only when volatile salts are mobile phase components. On the other hand there is a significant problem is the lack of sufficient selectivity for the most polar nucleosides and nucleotides. RP HPLC MS is still most often used in the determination of nucleosides and nucleotides, due to its high sensitivity and a comprehensive qualitative analysis. Another system used for the HPLC analysis of oligonucleotides is Ion Pair Reversed Phase High Performance Liquid Chromatography (IP RP HPLC). These compounds can not be analyzed by RP HPLC due to their high polarity. The advantage of IP RP HPLC is selectivity, achieved by a suitable choice of mobile phase composition and the possibility of using MS. A disadvantage of IP RP HPLC in the analysis of oligonucleotides is however lower sensitivity compared to RP HPLC. During the last few years Hydrophilic Interaction Liquid Chromatography (HILIC) was applied for the separation of mixtures of nucleosides, nucleotides, oligonucleotides extracted from a biological or food samples. The presented results demonstrate the usefulness of this method, however, the resolving power is limited due to the asymmetric peak shape. On the other hand proper selection of the mobile and stationary phase can lead to a high selectivity in the analysis of the most polar nucleosides, nucleotides and oligonucleotides, which can not be separated by RP HPLC.