Sugar beet molasses represents the most important by-product in the sugar beet industry which can be valorised through various methods due to very valuable composition. Standard beet molasses composition consists of sugar, nonsugar components and water. Furthermore, beet molasses normally contains betaine in the range from 3 to 8%/DM. Betaine is an important nonsugar component which is not eliminated during the production of sugar, so it accumulates in the molasses and could be obtained through separation process on an industrial chromatographic columns. The main objective of this research was to increase betaine content in betaine fractions and lower other impurities by applying different pretreatments before final separation process. Samples composition was characterized by using the HPLC technique and the quantification was performed by applying external standard method with betaine as a standard. Separation techniques used in this research were: (I) separation method without pretreatment (II) separation with preceded acidification (samples pH value adjustment) and (III) separation with alcohol fermentation as previous treatment. Betaine content in the starting sample was 62.76%/DM. The highest increase in betaine content was achieved by using separation method without pretreatment having final betaine content of 85.16%/DM. By using acidification as pretreatment before centrifugation the level of betaine was increased up to 67.68%/DM, whereas implementing the alcohol fermentation before centrifugation showed no significant differences compared to the starting sample.