An approach for rapid quantitation of 5-hydroxymethylfurfural (HMF) in honey using planar chromatography is suggested for the first time. In high-performance thin-layer chromatography (HPTLC) the migration time is approximately 5 min. Detection is performed by absorbance measurement at 290 nm. Polynomial calibration in the matrix over a range of 1:80 showed correlation coefficients, r , of ≥ 0.9997 for peak areas and ≥ 0.9996 for peak heights. Repeatability in the matrix confirmed the suitability of HPTLC–UV for quantitation of HMF in honey. The relative standard deviation ( RSD , %, n  = 6) of HMF at 10 ng/band was 2.9% (peak height) and 5.2% (peak area); it was 0.6% and 1.0%, respectively, at 100 ng/band. Other possible detection modes, for example fluorescence measurement after post-chromatographic derivatization and mass spectrometric detection, were also evaluated and can coupling can be used as an additional tool when it is necessary to confirm the results of prior quantitation by HPTLC–UV. The confirmation is provided by monitoring the HMF sodium adduct M + Na + at m / z 149 followed by quantitation in TIC or SIM mode. Detection limits for HPTLC–UV, HPTLC–MS (TIC), and HPTLC–MS (SIM) were 0.8 ng/band, 4 ng/band, and 0.9 ng/band, respectively. If 12 μL honey solution was applied to an HPTLC plate, the respective detection limits for HMF in honey corresponded to 0.6 mg kg −1 . Thus, the developed method was highly suitable for quantitation of HMF in honey at the strictest regulated level of 15 mg kg −1 . Comparison of HPTLC–UV detection with HPTLC–MS showed findings were comparable, with a mean deviation of 5.1 mg kg −1 for quantitation in SIM mode and 6.1 mg kg −1 for quantitation in TIC mode. The mean deviation of the HPTLC method compared with the HPLC method was 0.9 mg kg -1 HMF in honey. Re-evaluation of the same HPTLC plate after one month showed a deviation of 0.5 mg kg −1 HMF in honey. It was demonstrated that the proposed HPTLC method is an effective method for HMF quantitation in honey. Figure Fast quantitation of 5-hydroxymethylfurfural in honey