Liquid chromatography-mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple "dilute-and-shoot" approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences that lack consideration in the relevant metabolomics literature. In this work, we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-l-alanine-l-proline betaine (l,l-TMAP) and N,N-dimethyl-l-proline-l-proline betaine (l,l-DMPP), the latter reported here for the first time. Offline LC-MS/MS, magnetic resonance mass spectrometry (MRMS), and nuclear magnetic resonance (NMR) spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the coexistence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of column temperature and mobile-phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.