Purpose <p>Identification and quantification of sulfide ion in biological samples are required in forensic purpose. Gas chromatography-mass spectrometry (GC/MS) has been used for the analysis of sulfide ion by using derivatization reagents. However, conventional derivatization reagents require special attention for derivatization. To simplify the derivatization protocol, we examined ethenesulfonyl fluoride (ESF) as a derivatizing reagent of sulfide ion.</p> Methods <p>To 100 μL of whole blood sample containing sulfide ion, 100 μL of boric acid buffer (pH 8.0), 100 μL of acetone solution containing internal standard, 100 μL of acetone solution containing 600 mM concentration of ESF, and 100 μL of hexane were added in a 1.5-mL plastic tube. The mixture was vortexed at room temperature, the tubes were centrifuged, and the organic layer was injected into the GC/MS.</p> Results <p>ESF exhibited higher reactivity toward sulfide ion than interfering compounds present in whole blood, allowing for selective derivatization. With the optimized protocol, the detection limit for sulfide ion was 0.01&#xa0;μg/mL. The calibration curve showed good linearity (<i>R</i>² = 0.9999) in the range of 0.05–10.0&#xa0;μg/mL, and the precision (% relative standard deviation) and the accuracy (% bias) were within ± 10% (intra- and inter-day).</p> Conclusion <p>This GC/MS-based method is a valuable tool for forensic investigations and various analytical fields, offering reliable quantification of sulfide ion in whole blood.</p>