Since 2020, our lab has received blood samples from traffic cases involving suspicion of driving under the influence of nitrous oxide (N2O). While N2O analysis by gas chromatography (GC) has been around for decades, quantitative results in blood from drivers have been only scarcely reported. We present a three-year (2020–2022) retrospective study of N2O from traffic cases in Eastern Denmark with suspected involvement of N2O intake. Whole blood samples from traffic cases were analysed for N2O using headspace-GC-MS. Freshly made calibration curves and additions of xenon gas as an internal standard were used for calculation of N2O concentrations. Positive samples have been defined as having concentrations greater than 0.1 mL N2O/L blood. Over a three-year period, we have tested 62 traffic case blood samples for the presence of N2O. Despite the technical challenges associated with the analysis of N2O, we have found N2O in 52 of the samples. Calculated concentrations were in the range 0.1–48 mL N2O/L blood, which are similar to the few cases previously found in the literature.