Pyrrole (Py)-imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py-Im polyamide in rat plasma. Py-Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 microm, 2.1 x 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10-1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py-Im polyamide after intravenous administration.