Background/Objectives: Semaglutide, a long-acting glucagon-like peptide-1 (GLP-1) analog for type 2 diabetes and obesity, requires sensitive and high-throughput bioanalytical methods to support pharmacokinetic studies. However, previously reported liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays have been limited by lengthy run times (~18 min) and suboptimal sensitivity. This study aimed to develop and validate a rapid, sensitive LC-MS/MS method for quantifying semaglutide in plasma. Methods: Plasma samples (50 μL) were prepared by acetone-mediated protein precipitation followed by solid-phase extraction. Chromatographic separation was performed on a Cadenza CD-C18 MF column within 9 min, using positive electrospray ionization in multiple reaction monitoring mode with the transitions m/z 1029.4 → 110.1 for semaglutide and m/z 938.9 → 109.9 for liraglutide (internal standard). Validation followed the U.S. Food and Drug Administration (FDA) bioanalytical guidelines. Results: The assay showed a lower limit of quantification of 1 ng/mL with linearity across 1-500 ng/mL (R2 = 0.9999), with sharp peak shape and no carryover. Intra- and inter-day accuracies were 95.69-103.76% and 94.93-100.08%, with precision ≤4.50% and ≤5.88%. Recovery (93.05-107.95%) and matrix effects (96.34-104.12%) were consistent across quality control levels, and the analyte was stable under all tested conditions. The method was successfully applied to a pharmacokinetic study in Sprague-Dawley rats following subcutaneous administration of 50 μg semaglutide. Conclusions: The validated method offers shorter analysis time, improved sensitivity, and reduced sample volume compared with previously reported assays, supporting its application in preclinical pharmacokinetic studies of semaglutide and related GLP-1 analogs.
