Aim: We aimed to study the changes in osmolality of pasteurized donor human milk (PDHM) after the addition of four human milk fortifiers (HMFs), medium-chain triglyceride (MCT) oil, minerals, and vitamin supplements commonly used in neonates. Methods: The osmolality of 25 ml of PDHM was measured after the addition of 1 g each of HMF and 0.5 ml of MCT oil separately with a calibrated osmometer. The osmolality of 5 ml of PDHM was measured after the addition of minerals and vitamin supplements in therapeutic doses separately. All these measurements were carried out at 10 min, 30 min, and 2 h by a technician who was blinded to the fortifiers, minerals, and vitamin supplements used. Results: The osmolality of PDHM was 256 mOsm/kg (range = 256–259 mOsm/kg), which increased with all four HMFs tested, but remained below 450 mOsm/kg as recommended by the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN). Proprietary fortifiers increased the osmolality of PDHM to a maximum of 429 mOsm/kg (range = 306–429 mOsm/kg). However, there was no change in the osmolality with MCT oil. A maximum increase in osmolality was observed with the addition of calcium phosphate syrup to PDHM (1181 mOsm/kg). The addition of multivitamins and Vitamin D3 in therapeutic doses increased osmolality (range = 552–753 mOsm/kg) beyond the safety limits of ESPGHAN recommendation. Conclusions: HMFs increased the osmolality of PDHM but were within the safety limits of ESPGHAN recommendation. MCT oil did not affect the osmolality of PDHM. Minerals and vitamin supplements added in therapeutic dosages significantly increased the osmolality of PDHM. Minerals and vitamin supplements need to be appropriately diluted to keep osmolality below 450 mOsm/kg to ensure safety. Future research should focus on manufacturing fortifiers and supplements with low osmolality to ensure safety.