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A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2M hydrogen chloride (HCl) to plasma before SPE for the stability of WKYMVm peptide. Then, sample preparation using micro-elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration2), with an equation y=ax2+bx+c, was used to fit calibration curves over the concentration range of 3.02~2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25 % accuracy and precision values. For quality control samples at 15, 165, and 1820 ng/mL from the quantification experiment, the within-run and the between-run accuracy ranged from 92.5 to 123.4 % with precision values ≤15.1 % for WKYMVm peptide from the nominal values. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.