Probenecid (PB), a drug employed to reduce the excretion of other drugs in urine and also employed as masking agent in sport doping, is characterized for the first time in this work by vibrational spectroscopy. Previously, FTIR and FT-Raman spectra were assigned on the basis of density functional theory calculations. Afterwards, a detection protocol of PB was developed on the basis of surface-enhanced Raman scattering (SERS) spectroscopy. To accomplish this, the experimental conditions of SERS measurement were previously optimized by varying parameters such as the metal, its reduction protocol to fabricate the nanoparticles and the pH. The adsorption isotherms of PB on the metal nanoparticles were obtained, and the adsorption mechanism of this drug on Ag nanoparticles was deduced from the analysis of SERS spectra. Finally, a quantitative SERS study was carried out on the basis of the calibration curve deduced from the adsorption isotherms. The results shown in this study demonstrate that PB can be detected by SERS at concentrations as low as 1.2 µg/ml. Copyright © 2013 John Wiley & Sons, Ltd.