An automated on-line dialysis coupled to a trace enrichment method has been developed for the separation and quantification of four methylenedioxylated amphetamines in serum and plasma, using liquid chromatography coupled to a fluorimetric detector. The on-line dialysis method was optimized and validated on fresh human serum and plasma samples. This sample preparation method allowed the quantification of methylenedioxylated amphetamines in serum or plasma, at concentrations as low as ca. 10 ng ml-1, with good repeatability, reproducibility and accuracy. The automated on-line dialysis method took less than 30 min. This method was applied to seven toxicological cases and results showed that the concentration of methylenedioxylated amphetamines in blood was in the range of 20-484 ng ml-1.