OBJECTIVES: In this study, we aimed to establish a method for quantifying bromide ions (Br- ) in blood and urine using gas chromatograph-mass spectrometer (GC-MS) equipped with a headspace sampler, for biological monitoring of workers exposed to methyl bromide. METHODS: Samples were mixed with dimethyl sulfate, and Br- ions were detected using GC-MS with a headspace sampler. The validity of the proposed method was evaluated based on most of the US FDA guidance. The values obtained were compared with reference values by analysis using SeronormTM Trace Elements Whole Blood L-1 RUO. RESULTS: The calibration curve showed good linearity in the Br- concentration range of 0.1-20.0 mg/L, and the coefficient of determination R2 value was >.999. Intraday and interday accuracy values were 99.3%-103.1% and 97.4%-101.8%, respectively. The measured and reference values of Seronorm were concordant. Herein, eight urine and serum samples of workers were analyzed; the samples' Br- concentrations were known. The correlation coefficients of urine and serum samples were 0.97 and 0.96, respectively, and results were consistent. CONCLUSIONS: This study established a simple and rapid method for the determination of Br- concentration in biological samples using GC-MS with a headspace sampler. Moreover, it can be used for biological monitoring of occupational exposure to methyl bromide and for the determination of Br- concentration in a wide range of biological samples.