ABSTRACT An ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for the simultaneous determination of fipronil and its three metabolites (fipronil sulfone, fipronil sulfide, and fipronil desulfinyl) in zebrafish tissues. Protein precipitation was employed for sample preparation, followed by chromatographic separation on a C 18 column. The method demonstrated excellent linearity ( r 2 > 0.99) across a range of 0.1–10 ng/mL, with a lower limit of quantification (LLOQ) of 0.1 ng/mL for all analytes. Validation results confirmed acceptable accuracy (−5.0% to 8.9%), precision (0.6%–13.2%), recoveries (86.3%–113.6%), and minimal matrix effects (86.0%–110.6%). When applied to zebrafish exposed to 50 µg/L fipronil for 12 h, the method revealed preferential accumulation of fipronil and fipronil sulfone in liver tissues, while lower levels were detected in muscle tissues. Fipronil sulfide was rarely detected, and fipronil desulfinyl concentrations were below the LLOQ (0.1 ng/mL) in all tested samples. This reliable and sensitive method is well‐suited for environmental toxicology studies, pesticide residue monitoring, and ecological risk assessment.