Exenatide is the first approved glucagon-like peptide 1 receptor agonist subcutaneously or intramus-cularly injected for the treatment of type 2 diabetes mellitus. Typical therapeutic plasma concentrations are in the low pg/mL range, therefore requiring ultra-sensitive quantification. To enable the accurate evaluation of pharmacokinetic studies, we established a UPLC-MS/MS assay with a lower limit of quantification (LLOQ) of 5 pg/mL (1.2 pM) using 200μL of plasma, validated according to FDA''s and EMA''s pertinent guidelines. Exenatide was isolated from plasma with solid phase extraction utilizing anion-exchange sorbent. Quantification was performed with positive electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The calibrated concentration range of 5-10,000 pg/mL was linear showing correlation coefficients>0.99. Interday and intraday accuracy ranged from 97.5％to 105.4％with corresponding precision of<10.9％. Accuracy at the LLOQ ranged from 93.0％to 102.5％with corresponding precision of<15.9％. Because of the validity of a 10-fold dilution QC (accuracy 111.2％), the assay is suitable for exenatide quantification up to 100,000 pg/mL. The ultra-sensitive assay''s applicability was demonstrated by the quantification of exenatide plasma concentrations and pharma-cokinetics after intravenous and nasal administration to beagle dogs.