The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.