An untargeted metabolomics approach was utilized to determine urinary metabolites that could serve as small-molecule biomarkers for treatment response to standard tuberculosis treatment. However, the majority of metabolites that most accurately distinguished patient samples at the time of diagnosis from those at 1 month after the start of therapy lacked structural identification. The detection of unknown metabolite structures is a well-known limitation of untargeted metabolomics and underscores a need for continued elucidation of novel metabolite structures. In this study, we sought to define the structure of a urine metabolite with an experimentally determined mass of 202.1326 Da, classified as molecular feature (MF) 202.1326. A hypothesized structure of N¹-acetylisoputreanine was developed for MF 202.1326 using in silico tools and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the absence of a commercial standard, synthetic N¹-acetylisoputreanine was generated using enzymatic and chemical syntheses, and LC–MS/MS was used to confirm the structure of MF 202.1326 as N¹-acetylisoputreanine, a proposed terminal polyamine catabolite that had not been previously detected in biological samples. Further analysis demonstrated that N¹-acetylisoputreanine and an alternative form of this metabolite, N¹-acetylisoputreanine-γ-lactam, are both present in human urine and are likely end-products of polyamine metabolism.