New psychoactive substances (NPS)-designed to mimic various legal or illegal substances-are an emerging worldwide health problem. Their identification and quantification in either complex seized samples or powders are critical; moreover, their determination in biological fluids is an intriguing goal in the forensic toxicology field. Synthetic cathinones are one of the most important groups among NPS. The current paper was designed as a pilot study to investigate the application of NMR techniques to identify and quantify unknown NPS compounds in deuterated dimethyl sulfoxide (DMSO-d) and in urine using the synthetic cathinone benzylone (3,4-methylenedioxy-N-benzylcathinone, BMDP) as a pilot compound. In the first part of our study, nuclear magnetic resonance (NMR) spectroscopic characterization was performed using 1D and 2D homonuclear and heteronuclear NMR spectroscopic methods as long as diffusion ordered spectroscopy (DOSY). Following the above, the assignment of benzylone in DMSO-d was performed, and a distinct spectroscopic pattern was proposed. In the second part of our study, a NMR spectroscopic approach was applied for benzylone identification and quantification in a spiked with benzylone urine. Following the above, the assignment of benzylone in spiked urine was performed. A distinct pattern of the H11, H14, H15, and H8 signals on the H NMR spectra was observed and suggested as a "NMR spectroscopic pattern/signature" enabling the identification of benzylone moieties in urine. On the other hand, the applied NMR techniques showed low sensitivity in quantitating benzylone in spiked urine. Overall, our results are promising in using NMR for structure determination of unknown compounds in urine.