Abstract: Plasma (1.26 ml) was introduced into a water air-segmented donor stream (0.42 mL min-1) and passed through a dialysis membrane into a non-segmented water stream (0.6 mL min-1). The acceptor stream was concentrated on a column (12 x 2 mm) of Polysil C8 (63 µm). The trapping column was subsequently desorbed for online separation of rogletimide (I) by HPLC on a column (10 cm x 4.6 mm) of Nucleosil C8 (5 µm) with a mobile phase of 30% methanol in 100 mM ammonium acetate and MS or MS - MS detection. Gluthetimide was used as an internal standard. The calibration graph was rectilinear from 5 (determination limit) to 360 ng mL-1 of I and the coefficient of variation was 5.2% (n = 5) at the 100 ng mg-1 level. The application of a continuous-flow dialysis system, consisting of a membrane dialyzer and a trace enrichment column, in online combination with tandem mass spectrometry via a thermospray interface is described. The method is applied to the quantitation of drugs in complex biological matrixes containing macromol. interferences. The potential of the method is demonstrated by the quant. anal. of the anti-cancer drug rogletimide in the plasma of patients after treatment.