The growing interest for high quality solubility data in the early stages of drug discovery suggested a detailed optimization of experimental conditions for a 96-well HTS UV method in order to obtain solubility values close to thermodynamic solubility measured by shake-flask method. Results have shown that solubility data obtained by the HTS approach were highly dependent on shaking intensity and incubation times due to the formation of supersaturated solutions resulting from the dilution of DMSO stock solutions in aqueous buffer. Thus, careful experimental set-up was developed to improve the quality and the reproducibility of the HTS method. Moreover, the early qualitative prediction of bioavailability and absorption of orally administered drugs require more and more biorelevant solubility values in drug discovery programs. Thus, the optimized HTS method was also adapted to measure solubility directly in FaSSIF and FeSSIF media. The versatile HTS UV approach presented in this paper provides a unique and reliable way to determine solubility in various experimental conditions.