Abstract A reversed-phase, ion-pair high-performance liquid Chromatographic (HPLC) method for the determination of gallamine and its impurities is described. The separation is achieved on a Nucleosil C 18 column with acetonitrile-aqueous phosphate buffer (pH 3.0) (31:69, v/v) containing 0.1 M sodium perchlorate as eluent and on-line UV detection at 200 nm. The method is sensitive (the detection limit is 0.7 ng injected) and reproducible, with a peak area coefficient of variation of 0.19% ( n = 15; 3 μg injected) and 1.65% ( n = 15; 10 ng injected) for a gallamine assay; the detector response is linear over the concentration range 0.5–250 μ g/ml of gallamine triethiodide with a correlation coefficient of 0.9997. The method has been used to isolate the two main impurities contained in gallamine triethiodide batches; their structures have been determined by NMR and fast atom bombardment mass spectrometry. Various gallamine triethiodide batches have been analysed and the HPLC results compared with those obtained by thin-layer chromatography.