Rhodamine B and its five de-ethylated transformation products could be identified in a groundwater sample. Using high-performance thin-layer chromatography (HPTLC) six fluorescent zones were detected in the sample. In order to identify the compounds in the zones by exact mass mass spectrometry (MS) measurements and tandem mass spectrometry (MS/MS), they were extracted from the HPTLC plate for subsequent analysis by nano-chip high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (nano-chip HPLC/QTOFMS). In addition, chemical derivatisation experiments on HPTLC plates were applied to detect the presence of a primary amino group in the transformation products. From the combined analytical results it was possible to allocate rhodamine B and its five de-ethylated transformation products to the six different HPTLC zones. The quantification of rhodamineB indifferent groundwatersamples was carried out bya high-performance liquid chromatography/triple quadrupole mass spectrometry (HPLC/MS/MS). The maximum detected concentration of rhodamine B was 83μgL ^-1.