Controlling glucose content in potato tubers is a key challenge at two critical stages of the supply chain: (1) pre-harvest, to determine the optimal harvest date when glucose content is minimal and starch accumulation is maximal; (2) during post-harvest storage, to detect cold sweetening early and initiate preventive thermal reconditioning. This study reports the full validation of an enzymatic assay method based on the glucose oxidase–peroxidase (GOD/POD) system, implemented on the Gluco-LIS® portable reader, in accordance with ICH Q2(R1) and ISO 5725 requirements, conducted at the laboratory of the Technical Center for Potatoes and Artichokes (CTPTA, Manouba-Essaïda, Tunisia). Nine performance parameters were evaluated over the 10–500 mg/dL range: specificity, linearity, accuracy, repeatability, reproducibility, limit of detection (LOD), limit of quantification (LOQ), robustness, and stability. The calibration curve has R² = 0.9999 (y = 1.0029x + 0.878 mg/dL). Recovery rates range from 99.2% to 106.9%, repeatability CVs remain below 3.1% for c ≥ 100 mg/dL, and reproducibility CVs do not exceed 3.5% in a 3-day × 2-operator cross-validation. The operational LOQ was experimentally confirmed at 10 mg/dL (CV=9.9%) and covers the whole range of glucose levels in stored tubers. Robustness, tested by variation of laboratory ambient temperature (±2°C), does not show a significant effect (F = 3.158; p = 0.079). The stability protocol has been adapted because of the intrinsic enzymatic instability of raw juice (browning with polyphenol oxidase - PPO), confirming the short-term chemical stability of D-glucose solution at 22°C (H0-H3) and under refrigeration at 4°C (J0-J3). These results validate Gluco-LIS® as a field tool for real-time post-harvest carbohydrate quality monitoring for potatoes following ICH Q2(R1).