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 (4.1-2030 nM) with a limit of detection (LOD) of 4.1 nM N(2)O (18 ppb) and a limit of quantification (LOQ) of 16 nM N(2)O (72 ppb). 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.