A validated method for the determination of triethylenetetramine, a selective copper-chelator currently undergoing clinical trials for the treatment of diabetic heart failure, and its two major metabolites, N(1)-acetyltriethylenetetramine and N(1),N(10)-diacetyltriethylenetetramine in human serum using HPLC is reported. The method used 9-flouorenylmethylchloroformate chloride to label all three analytes. The fluorescence labeled analytes were then separated chromatographically using a reversed phase C18 column under a gradient elution program and detected spectrofluorometrically at 317 nm with excitation at 263 nm. Application of the method is demonstrated by pharmacokinetic measurement in one healthy volunteer taking the drug orally.