In this study, a sensitive, accurate and cost-effective ultra-performance liquid chromatography-tandem mass spectrometric method was developed using a new clean-up process for the quantification of leuprolide in rat plasma. Chromatography was carried on a HALO peptide ES-C18 column using water and acetonitrile (containing 1% acetic acid) as the mobile phase with a gradient elution. Electrospray ionization was applied and operated in the multiple reaction monitoring positive mode. Leuprolide and an internal standard (leuprolide-d5) were extracted by the liquid？liquid extraction with methylene chloride after protein precipitation by the new clean-up process. In the clean-up process, acetic acid was used for higher reproducibility, and then 10 lL of cleaned aqueous phase was finally injected into the UPLC？MS/MS system. The chromatograms showed good resolution, and no interfering peaks to plasma constituents were observed. The lower limit of quantification in rat plasma was 20 pg/mL for leuprolide. The calibration curves for leuprolide in rat plasma were linear over all the concentration range 20？10,000 pg/mL with a correlation coefficient of 0.991. The intra- and inter-day assay for precision and accuracy fulfilled the international guideline requirements.