Characterization of N,N 0 -substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N-diand tri-alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N 0 -substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies.Methods: High-resolution electrospray ionization tandem mass spectrometry (ESI-HR-MS/MS) has been used to characterize a series of N,N 0 -substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired