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Background: Mother's own milk does not provide enough nutrients to feed a preterm baby born before 32 weeks' gestation; therefore, human milk fortifiers are needed. However, human milk fortifiers increase the osmolality, and enteral administration of high osmolality fluids has been associated with gastrointestinal symptoms. For this reason, it is necessary for laboratories to have a validated system in order to measure human milk osmolality., Research Aim: The aim of this study was to validate the OM-6050 Station System for measuring the osmolality of fortified mother's milk samples., Methods: Osmolality was measured using the osmometer OM-6050 Station System. Milk samples from healthy mothers ( N = 3) unfortified and with two fortifiers (Almirón Fortifier ® or NAN FM85 ® ), as well as a nutritional supplement (Duocal MCT ® ) were used in the validation study through precision and linearity analysis., Results: In the precision study the mean intra-assay coefficient of variation was 1.2% and 1.7% for mother's milk and fortified mother's milk, respectively. The mean inter-assay coefficient of variation was ≤ 1% in both cases. In the linearity study the regression analysis had a linear response to fortified mother's milk osmolality between 294 mOsm/kg and 539 mOsm/kg., Conclusion: The osmometer OM-6050 Station was reliable for determining the osmolality of fortified and unfortified mother's milk. It may be useful in the clinical practices within Neonatal Intensive Care Units.