We obtained monoclonal antibodies against N¹,N¹²-diacetylspermine (DiAcSpm) and N¹, N⁸-diacetylspermidine (DiAcSpd), and developed two systems of competitive ELISA that utilize the antibodies and a common enzyme-labeled antigen to measure these diacetylpolyamines. Cross-reactions with N¹-acetylspermidine in the assay of DiAcSpm and with N⁸-acetylspermidine in the assay of DiAcSpd were as low as 0.26 and 0.6%, respectively, and were judged to be insignificant in clinical use for measuring urinary diacetylpolyamines. These assays were used to assess diurnal variations in diacetyl-polyamine excretion in urine to show that the excretion of diacetylpolyamines after normalization for the concentration of creatinine is stable over a day with only minimal diurnal variation.