N-Acetylglucosamine is an ingredient in pharmaceuticals, nutritional supplements and in cosmetics. N-Acetylglucosamine in cosmetics is expected to improve skin hydration, reparation, and to contribute as anti-wrinkle agent. This study reports on the validation and application of an HPLC method based on HILIC and UV detection for determining N-acetylglucosamine in cosmetics and in samples obtained after testing the skin exposed to cosmetics formulations. The chromatographic column used is a ZIC(®)-pHILIC (150 mm × 4.6 mm, 5 μm particle size) on which a mobile phase containing acetonitrile-aqueous KH2PO4 (70:30, v/v) 15 mM was applied in isocratic elution mode injecting 20 μl of sample at 0.5 ml/min constant flow-rate and 10±1°C column temperature. Under these conditions the total run time was 10 min and N-acetylglucosamine eluted baseline separated from all other compounds in the samples. Calibration in the range from 40 to 80 μg/ml allowed to assess the method linearity (R(2)>0.999) in a concentration range corresponding to about 50% to 120% of the expected levels of N-acetylglucosamine in the formulations. Precision expressed by RSD% was always better than 2% in intra-day and inter-day assays of authentic samples. Accuracy was in all cases within 95-105% of the expected concentration value in formulations containing N-acetylglucosamine. The sensitivity of the method was at the level of 10 μg/ml as limit of detection, and at 40 μg/ml as limit of quantitation. The application of the method to formulations containing solid lipid nanoparticles documents its usefulness in cosmetic quality control. The results witness that the method is also suitable for the determination of N-acetylglucosamine in samples obtained from skin test strips.