A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C18 column with a mobile phase of acetonitrile-0.05 M KH2PO4 (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1-3.4% and 1.9-2.4% for all analytes and the accuracy was 101-107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I-IV in clinical pharmacokinetic investigations.