A variety of factors have been recognized that influence media optimization for drug release studies of implant dosage forms. Of primary importance is selection of a medium that physiologically mimics the milieu at the site of administration (a condition termed ‘biorelevance’). We describe in this paper, the characterization of Hanks’ balanced salts solution, with necessary modification, for application as a ‘biorelevant’ medium for in vitro release studies of a biodegradable, subcutaneous implant of naltrexone. A detailed investigation of changes in pH, osmolality and ultraviolet (UV) spectrum as a function of time and temperature was conducted. Variation in the parameters evaluated was found to be within acceptable limits. Validation of a simple and selective, high performance liquid chromatography (HPLC) assay method for naltrexone was carried out to evaluate stability. The calibration curves were linear from 0.16 to 20.00 μg ml−1. Imprecision and inaccuracy were less than 2% and no interference was observed from degradation peaks. Stability studies of naltrexone indicated the media should be replaced every 7–8 days for real-time testing. This was applied to an investigation of in vitro drug release. The method has been proven to be suitable for investigation of naltrexone released from the implant.