A sensitive, accurate and specific high-performance thin-layer chromatographic method has been developed for determination of nafazatrom, a pyrazolinone derivative with antithrombotic and antimetastatic activity. Because of the instability of the drug during clean-up procedures, nafazatrom is recovered from plasma and urine with a single extraction and the extract is spotted directly onto the chromatographic plate. The specificity of the assay was increased by post-chromatographic derivatization with 4-dimethylaminobenzaldehyde. Up to eight primary metabolites with an unchanged position 4 in the heterocyclic ring were also detected and separated chromatographically. The detection limit for nafazatrom is 0.5 ng per spot and 5 ng/ml. The analytical error in the nanogram per millilitre range is less than 10%, low enough for pharmacokinetic studies and for plasma level monitoring in patients.