Alpibectir (also known as BVL-GSK098 and GSK3729098) is a new chemical entity with a novel mechanism for the treatment of tuberculosis. The disposition of alpibectir was determined in subjects from a first in human trial after a single oral dose of 40 mg, and after 7 days repeat dosing at 30 mg. Here we present a combined approach of UPLC-HRMS, 1H and 19F-NMR to comprehensively determine the human metabolic fate of alpibectir. Utilising multiple sites of fluorination in the molecule, it was possible to fractionate clinical urine and plasma to confidently detect and quantify the metabolite responses using 19F-NMR. Qualitative detection and structural characterisation of F-containing NMR fractions was complemented by UPLC-MS/MS to further add confidence to the metabolite responses in these fractions. Subsequent 1H NMR then provided unequivocal standard-free structural confirmation for key metabolites, which would not be possible with conventional radioactivity detection and LC-MS/MS techniques. Alpibectir was shown to undergo extensive hydrolysis of its central amide moiety. Downstream biotransformations of both hydrolytic products, to trifluorobutanoic and trifluoroacetic acid and the product of carbamate conjugation respectively, were detected initially by unbiased 19F-NMR detection. The qualitative metabolic profile and quantitative determination of the percentage of dose excreted in urine was delivered. Significance Statement This study demonstrates that composite NMR and MS datasets help provide more complete data rationalisation and a confident assessment of human metabolism, and thereby has permitted early risk assessment and progression of activities relating to qualification of these risks for alpibectir. By combining the sensitivity of UPLC-HRMS, the selectivity of 19F-NMR, and structure-rich nature of 1H-NMR, an alternative approach for the detection and quantification of the drug-related material compared to traditional human ADME profiling using radiolabelled drug can be considered.