The environmental persistence and structural diversity of per- and polyfluoroalkyl substances (PFAS) complicate their multimedia monitoring. This complexity poses a substantial analytical challenge, necessitating reliable methods for the simultaneous quantification of multiple PFAS congeners. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of 38 PFASs. The chromatographic gradient elution conditions and mass spectrometry parameters, specifically collision energy (CE) and cone voltage (CV), were optimized to improve the chromatographic separation and ionization efficiency of individual analytes. Quantification was performed using a stable isotope-labeled internal standard strategy to ensure accuracy. High sensitivity was achieved, with detection limits ranging from 0.002 to 0.14 μg/L. Recovery rates for most compounds at trace-level concentrations ranged from 90% to 130%, meeting rigorous analytical criteria. The internal standard correction strategy further ensured quantitative reliability. This method provides an efficient, high-throughput analytial tool for comprehensive PFAS screening in various environmental matrices.