A liquid chromatography-mass spectrometry (LC-MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC-MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 M Omega), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M+H]+ values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.