HPLC method with SPE preseparation step for determination of silybin, the main constituent of silymarin, in blood plasma was optimized. A C₁₈ sorbent for preseparation from acidified samples was used. Diastereomers of silybin in unconjugated form or total silybin are resolved by the method, the detection limit being 2 nmol/l and the limit of quantification 5 nmol/l per diastereomer. The method is suitable for silybin monitoring in human plasma after administration of drugs or food additives based on silymarin.