A separation system based on isotachophoresis is described for the cyanogenic glycoside linamarin in aqueous solution and in human urine. Isotachophoresis is a migration of a substance in an electric field, which is applied to a system of electrolytes of specific design. Detection is carried out by monitoring conductivity changes. However, for linamarin in urine, a preseparation procedure was necessary because of the high amount of electrolytes. This was performed by affinity chromatography on a silica sorbent column, with cyclohexyl as the functional group by which linamarin was retained. After elution from the column by methanol, a separation and quantitation of linamarin was possible by means of isotachophoresis. The method allowed determinations of urinary linamarin exceeding 100 microM, with a coefficient of variation of 13% at 500 microM.