The objective of the study was to evaluate the different headspace equilibration methods for the quantification of dissolved greenhouse gases in groundwater. Groundwater samples were collected from wells with contrasting hydrogeochemical properties and degassed using the headspace equilibration method. One hundred samples from each well were randomly selected, treatments were applied and headspace gases analysed by gas chromatography. Headspace equilibration treatments varied helium (He):water ratio, shaking time and standing time. Mean groundwater N2O, CO2 and CH4 concentrations were 0.024 mg N L−1, 13.71 mg C L−1 and 1.63 μg C L−1, respectively. All treatments were found to significantly influence dissolved gas concentrations. Considerable differences in the optimal He:water ratio and standing time were observed between the three gases. For N2O, CO2 and CH4 the optimum operating points for He:water ratio was 4.4:1, 3:1 and 3.4:1; shaking time was 13, 12 and 13 min; and standing time was 63, 17 and 108 min, respectively. The headspace equilibration method needs to be harmonised to ensure comparability between studies. The experiment reveals that He:water ratio 3:1 and shaking time 13 min give better estimation of dissolved gases than any lower or higher ratios and shaking times. The standing time 63, 17 and 108 min should be applied for N2O, CO2 and CH4, respectively. ; peer-reviewed ; Department of Agriculture, Food and Fisheries, Ireland - Research Stimulus Fund Programme (Grant RSF 06383); Department of Civil, Structural and Environmental Engineering, Trinity College Dublin