Ptaquiloside (PTA) is a carcinogenic compound naturally occurring in bracken ferns (Pteridium aquilinum). It is highly water soluble and prone to leaching from topsoil to surface and groundwaters. Due to possible human exposure via drinking water, PTA is considered as an emerging contaminant. We present a sensitive and robust method for analysis of PTA and its degradation product pterosin B (PtB) in groundwater. The method comprises two steps: sample preservation at the field site followed by sample pre-concentration in the laboratory. The preservation step was developed by applying a Plackett–Burman experimental design testing the following variables: water type, pH, filtering, bottle type, storage temperature, transportation conditions and test time. The best sample preservation was obtained by using amber glass bottles, unfiltered solutions buffered at pH 6, transported without ice, stored at 4^◦ C and analysed within 48 h. The recovery was 94% to 100%. The sample purification step had a pre-concentration factor of 250, and the recovery percentages of the entire method were 85 ± 2 (PTA) and 91 ± 3 (PtB). The limits of detection (LOD) of the full method were 0.001 µg L⁻¹ and 0.0001 µg L⁻¹ for PTA and PtB, respectively. The method enables sensitive monitoring of PTA and PtB in groundwater. Carcinogenic PTA was detected in one groundwater well (0.35 µg L⁻¹).