Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been synthesized for use in a sustained delivery system. The aim of the present report was to develop and validate a simple, sensitive and reliable LC–MS/MS method for the simultaneous determination of RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-CN chromatography column (2.1mm×100mm, 3.5μm) by isocratic elution using a 0.2% formic acid aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample preparation method employed 50μL of a plasma sample and liquid-liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v) as the extraction solvent.The proposed method was fully validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and stability. Good linearity was found within the range of 0.1–10.0ng/mL for both analytes (r>0.996). This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation in rats following a single intramuscular injection and biological conversion in vitro.