Analysis of glycan part of membrane glycoproteins has become an important analytical challenge because of its high importance in intercellular recognition and interactions of molecules with receptors on the cell surface. Numerous attempts to isolate membrane glycoproteins have encountered a number of obstacles due to the lack of reproducibility of the method or pollution with cytosolic glycoprotein. Therefore, the aim of this study was to confirm the efficacy and reproducibility of the method of isolation of membrane proteins with Triton X-114 and its purification with organic solvents for the purpose of analyzing the N-glycosylation. The analysis of N-glycans was performed with HILIC-UPLC chromatography. Used cells were from THP cell lines, but the repeatability was confirmed with human brain cells, white blood cells and lysosomes. In the method for purifying the isolated membrane proteins are used various organic solvents with which we got a protein precipitate. Organic solvents which have been used are acetone, combinations of acetone/methanol and chloroform/methanol, where the last combination came as most effective, although all three methods gave chromatograms with the same glycan profile. It was also found that the initial amount of cells affects the intensity of the chromatographic peak. To confirm the presence of glycan structure in the obtained samples, individual chromatographic peaks were collected and sequenced with combination of exoglycosidases that are tearing certain non-reducing ends of the monosaccharides from glycans. Thus were successfully proven and identified some glycan structures. The importance of this study is progress in isolation of membrane proteins thus providing a better insight into their N-glycosylation that is, except for the diagnosis and prognosis of disease, today very important in biopharmaceutical industry because glycosylation has an effect on the physical and chemical properties of the protein which is reflected in their effectiveness and safety.