PSTi8 is a 21 amino acid pancreastatin inhibitory peptide that demonstrated potent antidiabetic activity in insulin resistant rodent models. The goal of the current work is to establish and validate the LC-ESI-MS/MS bioanalytical assay of PSTi8 in mice plasma in order to unveil its pharmacokinetic (PK) behaviour for the first time. The MS detection of PSTi8 and diprotin A (internal standard, IS) was conducted with Q1/Q3 SRM transitions at 607.80 ([M+4 H])/771.20 and 342.20/229.10, respectively using positive ESI. Phenomenex Aqua 5μ 125A (250 × 4.6 mm) column was utilized to separate PSTi8 and IS with a mobile phase consists of MeOH-0.1 % formic acid (1:1, v/v) using 0.4 mL/min flow rate. SPE using medium anion exchange cartridge (Oasis MAX) was used for the extraction of analyte and IS from the mice plasma and the extraction recovery was found to be >55 %. PSTi8 displayed good linearity across the 5-1000 ng/mL concentrations range. The intra- and inter- day accuracy was observed between 99.44-110.20 % and 99.66-110.93 %, respectively. The intra- and inter- day precision was observed between 2.61-4.03 % and 2.90-7.16 %, respectively. The intra-day and inter-day accuracy and precision data was within the 100 ± 15 % nominal values recommended by the United States Food and Drug Administration bioanalytical guidance. The LC-MS/MS assay was validated effectively to investigate the PSTi8 plasma concentrations following intravenous and intraperitoneal PK studies in mice. The absolute bioavailability of PSTi8 was 52.74 ± 13.50 %.