1,4-Dimethylnaphthalene (1,4-DMN) is effective sprout suppressant used in potato stores in many countries in the world. High residue levels of this compound on the potatoes and in other environmental samples are considered for human health and environmental risks. Determination of the residue requires specific analytical methods to be developed and validated. In this study, HPLC-UV was selected for validating a separation method based on reversed phase for the analysis of 1,4-DMN using 2-methylnaphthalene (2-MeN) as internal standard testing three HPLC systems. Under the same chromatographic conditions, all three systems achieved good separation on a Jones column (Hypersil ODS 5 μm, 250 mm × 4.6 mm) at ambient temperature isocratically using 70% acetonitrile as mobile phase at a flow rate of 1.5 mL min−1, 20 μL injection volume, a run time of 10 min, and a detection wavelength of 228 nm. All three systems showed high precision, good linearity, and low limit of detection (LOD) and quantification (LOQ); particularly, the SpectraSERIES UV100-autosampler system offered lower values of LOD (0.001–0.004 μg mL−1) and LOQ (0.002–0.013 μg mL−1) for both compounds. This system can be used for the quantitative determination of 1,4-DMN residue in potato and environmental samples.