A SPME-GC–MS method was developed to quantify nitrous oxide (N 2 O) to evaluate denitrification rates. There is a need for this sensitive and definitive N 2 O detection method to accurately measure the soil and sediment ability to convert anthropogenic mineral nitrogen loads to N 2 through denitrification hence decreasing estuarine waterway pollution loading. This method is applied to measure denitrification, which is a major pathway for inorganic nitrogen removal, by incorporating the acetylene (C 2 H 2 ) block method on anaerobic assays. Currently, denitrification is largely measured using GCs fitted with TCD or ECD detectors. With a mean R 2 value of 0.996, the calibration curve spanned over three orders of magnitude (4.1–2030nM) with a limit of detection (LOD) of 4.1nM N 2 O (18ppb) and a limit of quantification (LOQ) of 16nM N 2 O (72ppb). This detection method was valid with less than 15% relative standard deviation (RSD) and error for middle and high quality control (QC) points and less than 20% for low QC points on three experimental days. Measuring N 2 O using SPME-GC–MS technology allows for confidence in identification, high sensitivity, reproducibility, and short run times.