The paper describes the analysis of biodiesel (BD) obtained by transesterification of sunflower oil with methanol. The system proposed is the reversed-phase high-performance liquid chromatography (RP-HPLC) operating on a C18 (250 x 4.6 mm) column with UV detection at 205 nm and gradient elution in both nonaqueous (NARP) and aqueous-organic (ARP) modes. The former separates the main classes of compounds from BD, while the latter allows the separation of individual components belonging to the same class. The method enables separation of fatty acid methyl esters (FAME), monoacylglycerols (MG), diacylglycerols (DG) and triacylglycerols (TG). The results obtained reveal that the proposed approach is suitable for monitoring the process of biodiesel synthesis.