BACKGROUND We present formulation and stability evaluation of a 2% (w/v) phenylephrine hydrochloride biocompatible eye drop solution, routinely prepared in hospital pharmacy under aseptic conditions, for retinal examination of neonates and premature infants. METHODS Eye drop solution was formulated by dissolution of phenylephrine hydrochloride and disodium hydrogen phosphate as buffering agent in sterile water for injection and sodium chloride for injection as isotonic agent. The previous solution was sterile filtered through under aseptic conditions, in an iso class 5 air quality clean room under horizontal laminar airflow hood. Physical stability (visual inspection, osmolality measurements), chemical stability (pH measurement, phenylephrine assay by liquid chromatography coupled with an ultra-high resolution and accurate mass) and sterility evaluation of phenylephrine eye drop solution stored at ambient temperature were studied during 60 days. CONCLUSIONS The formulated eye drop solution had a pH of 6.90±0.05 and an osmolality of 285±2 mOsm/kg. Throughout the 60 days study the solutions remained clear without any precipitation or color modification, sterility was maintained, pH and osmolality were not significantly modified and no significant loss of product was detected using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument suggesting the lack of degradation. CONCLUSIONS These results indicate that 2% phenylephrine hydrochloride eye drop solutions were physically, chemically and microbiologically stable for at least 60 days when stored in type I amber glass vials at room temperature, allowing the compounding of higher batch sizes.