Abstract Liquid organic hydrogen carriers (LOHC) are an interesting option for hydrogen storage and transportation. This concept is based on reversible hydrogenation and dehydrogenation of a carrier compound for uptake and release of hydrogen respectively. Among others, dibenzyltoluene is a potential LOHC due to its reasonable hydrogen storage capacity (6.2 ma-%) and high thermal stability. However, a huge number of stable intermediates with different degrees of hydrogenation are observed in a partially hydrogenated reaction mixture of dibenzyltoluene. For the process development and studies of the dibenzyltoluene reaction mechanism, it is crucial to determine physico-chemical properties of its various partially hydrogenated fractions, which requires their isolation from the reaction mixture. In this work, a reversed-phase high performance liquid chromatography (RP-HPLC) method for the separation and purification of partially hydrogenated mixtures of dibenzyltoluene is presented. The method was developed and validated at analytical scale and successfully scaled up to semi-preparative scale. The mixture was separated into four fractions according to their degree of hydrogenations using phenylhexyl silica stationary phase and a mobile phase consisting of acetone/water (96/4, v/v). Fractions with purity above 98% and yield higher than 90% were obtained in a semi-preparative column with an internal diameter of 50 mm.