A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the high performance liquid chromatography determination of the content of 1-deoxynojirimycin (DNJ) in a new dietary supplement in the form of granules for oral solution preparation. The derivatization reaction was carried out at room temperature for 15 min at pH 7. The reaction reached completeness at a reagent to analyte molar ratio of about 60. The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion stainless steel column (250 mm × 4.6 mm; 4 µm) with detection at λ = 254 nm. The mobile phase consisted of triethylammonium (TEA) phosphate buffer (0.05 M; pH 3) and acetonitrile under gradient conditions at a flow-rate ramping from 1 to 1.2 mL/min. The validation parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were satisfactory. Intra-day precision (relative standard deviation, RSD) was ≤ 2.23% for peak area and retention time without significant differences between intra- and inter-day data. Recovery studies gave good results (93.59%; n = 15) with a RSD of 2.64%. The developed method is suitable for the quality control of DNJ in raw material and industrial products. The method can be applied in any analytical laboratory and does not require sophisticated instrumentation.