The evaluation of new tuberculosis (TB) therapies is limited by the paucity of biomarkers to monitor treatment response. Previous work detected an uncharacterized urine metabolite with a molecular mass of 874.3547 Da that showed promise as a biomarker for successful TB treatment. Using mass spectrometry combined with enzymatic digestions, the metabolite was structurally characterized as a seryl-leucine core 1 O-glycosylated peptide (SLC1G) of human origin. Examination of SLC1G in urine revealed a significant abundance increase in individuals with active TB versus their household contacts and healthy controls. Moreover, differential decreases in SLC1G levels were observed by week one in TB patients during successful treatment versus those that failed treatment. The SLC1G levels were also associated with clinical parameters used to measure bacterial burden (GeneXpert) and inflammation (positron emission tomography-computed tomography (PET-CT)). These results demonstrate the importance of metabolite identification and provide strong evidence for applying SLC1G as a biomarker of TB treatment response.