TAK-448 is a nonapeptide analogue and a novel metastin receptor agonist. The aim of this study was to develop a bioanalytical method for TAK-448F (the free base of TAK-448) in human plasma with LC/MS/MS that is sensitive and applicable for the clinical PK studies, and to evaluate the reliability and robustness of the developed method through a validation study in accordance with the regulatory guidance/guideline. The bioanalytical method developed in this study can be outlined as follows. The structural analogue, TAK-683, was used as the internal standard (IS). TAK-448F and the IS were extracted from human plasma using solid phase extraction (SPE) with a polymer-based weak cationic exchanger. After evaporating, the residue was reconstituted and injected into a LC–MS/MS system with ESI probe and analyzed by the selected reaction monitoring (SRM) in the positive ion mode. Separation was performed through an UPLC BEH Phenyl column with the mobile phase of water/methanol/formic acid mixture at a flow rate of 0.2 mL/min. The total run time was 10 minutes. The LLOQ was achieved to be 5 pg/mL with 0.5 mL of human plasma sample. All the validation results met the acceptance criteria in accordance with the regulatory guidance/guideline proving its reliability and robustness. As a result of the clinical study, the human PK profiles of TAK-448F were successfully obtained with this method.