We formerly developed and applied a liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry technique for the detection and identification of exogenous compounds in clinical and forensic toxicology. In this study, we aimed to adapt this technique to the detection and identification of the constituents of the urinary peptidome. After solid-phase extraction, separation was performed using gradient reversed-phase liquid chromatography. The mass spectrometer was operated in the information-dependent acquisition mode, switching between: a survey scan acquired in the enhanced multi-charged scan mode with dynamic subtraction of background noise; and two dependent scans obtained in the enhanced product ion scan mode. The results obtained show that: (i) the present procedure is able to detect and identify peptides which, together with their inferred parent proteins, are similar to those referenced in the related literature; (ii) the structure of some peptides can generally be resolved from their enhanced product ion spectra; and (iii) confirmation of the sequences proposed through library search by in silico verification of the fragments observed in the enhanced product ion spectra seems to be indispensable to avoid misinterpretations.