Increasingly, pharmaceutical and biotech companies have begun to realize the importance of obtaining solubility information in early drug discovery as it is one of the critical parameters for lead selection and optimization. This report introduces a high‐throughput equilibrium solubility (HT‐Eq sol) assay using a novel miniaturized shake‐flask approach and streamlined HPLC analysis. The new HT‐Eq sol assay, validated and optimized via a test set of 85 marketed drugs and Novartis internal compounds, shows an excellent correlation to the conventional shake‐flask thermodynamic solubility data generated in‐house and the equilibrium solubility results reported in literature. It therefore offers a fast, reliable and cost‐effective screening tool for solubility assessment in early drug discovery, allowing for prioritization of drug candidates using aqueous solubility in conjunction with other profiling information and efficacy data. Our work demonstrates that presence of a small amount of DMSO (0.5–5%) will result in significant overstimation of equilibrium solubility (up to 6 folds). In addition, monitoring of drug dissolution process using the current approach as well as the interplay between equilibrium solubility data and those from kinetic solubility are discussed. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 3052–3071, 2007