On the basis of a novel derivatization, a new planar chromatographic method has been developed for the determination of acrylamide (AA) in drinking water at the ultra-trace level. After SPE, the water extracts were oversprayed on a high-performance thin-layer chromatography (HPTLC) silica gel plate with the derivatization agent dansulfinic acid and derivatized in situ. Chromatography was performed with ethyl acetate and the fluorescent product was quantified at 366/>400 nm. Verification was based on HPTLC-ESI/MS, HPTLC-direct analysis in real-time (DART)-TOF/MS and NMR. The routine HPTLC-fluorescence detection (FLD) method was validated for spiked drinking water. The regression analysis was linear (r >0.9918) in the range of 0.1–0.4 μg/L. LOD was calculated to be 0.025 μg/L and experimentally proved for spiked samples at levels down to 0.05 μg/L (S/N = 6) which was suited for monitoring the EU limit value of 0.1 μg/L in drinking water (0.5 μg/L demanded by World Health Organization (WHO)/US Environmental Protection Agency (EPA)). Within-run precision and the mean between-run precision (RSD, n = 3, three concentration levels each) were evaluated to be 4.8 and 11.0%, respectively. The mean recovery (0.1, 0.2, and 0.3 μg/L) was 96% corrected by the internal standard. The method, in comparison with HPLC-MS/MS showed comparable results and demonstrated the accuracy of the method.