Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with known mutagenic and carcinogenic properties. They are commonly introduced into aquatic environments through industrial activities, fossil fuel combustion, and residential heating. Given their low concentrations and environmental relevance, the development of sensitive, selective, and cost-effective methods for their determination in water matrices is of critical importance. This study presents an optimized and validated analytical methodology for the simultaneous quantification of six priority PAHs—fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene—in drinking water, groundwater, and surface water. The method employs stir bar sorptive extraction (SBSE) followed by high-performance liquid chromatography with fluorescence detection (HPLC–FLD). Key extraction parameters, including sample temperature, extraction time, stirring rate, matrix composition, and desorption time, were systematically optimized. The method demonstrated excellent linearity (R² > 0.998) across all analytes. Limits of detection ranged from 0.04 to 0.18 ng/L, while limits of quantification ranged from 0.13 to 0.61 ng/L. Recovery values were within 82–108%, with intra- and inter-day precision (RSD) below 10%. The total chromatographic runtime was 24 minutes, followed by a 2-minute post-run. Measurement uncertainty was also evaluated according to ISO/IEC 17025 guidelines.The validated SBSE–HPLC–FLD method provides a reliable, sensitive, and efficient approach for the monitoring of PAHs in environmental water matrices, contributing to improved risk assessment and regulatory compliance.