AbstractPurpose: Liposomes composed of soy phosphatidylcholine (PC) have been widely used in artificial tears for the treatment of dry eye disease (DED) due to their ability to supplement the lipid layer of the ocular surface. The disadvantage of PC extracted from natural sources is the difficulty of standardizing its fatty acid composition. Therefore, the use of synthetic phospholipids such as 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and Dimyristoylphosphatidylcholine (DMPC) allows better characterization and standardization than those from natural sources, being of interest in the preparation of ophthalmic liposomal formulations for DED.Methods: Liposomal formulations comprised of DOPC (F1) and DOPC combined with DMPC (F2) were prepared according to the Bangham technique and dispersed in a borate‐trehalose buffer. Formulations were characterized in terms of pH, size, osmolarity and surface tension. In vitro tolerance was performed in human corneal epithelial cells and compared with soy PC liposomal formulation.Results: Both F1 and F2 synthetic liposomal formulations showed suitable properties for topical ophthalmic administration for DED such as hypotonicity (F1: 244.8 ± 0.74; F2: 252.5 ± 0.08 mOsm/L), neutral pH (F1: 7.52 ± 0.01; F2: 7.54 ± 0.02), low surface tension (F1: 24.53 ± 0.70; F2: 26.93 ± 0.49 mN/m) and a vesicle size of 150.8 ± 59.60 and 181.00 ± 49.83 nm for F1 and F2 respectively. Synthetic phospholipids outperformed soy PC liposomes regarding in vitro tolerance in a very sensitive corneal line, exhibiting cell viability values after 1 hour exposure of 88.71 ± 7.32% and 82.36 ± 8.84% for F1 and F2 respectively compared to soy PC liposomes (73.08 ± 8.74%). After simulated chronic exposure (4 h), cell viability of F1 (78.26 ± 1.65%) and F2 (79.08 ± 1.75%) were higher than the one obtained for PC liposomal formulation (64.53 ± 6.73%).Conclusions: Liposomes composed of synthetic phospholipids are potential candidates for the development of artificial tears for DED treatment.