Aqueous solubility is a critical physicochemical property and must be addressed early during drug discovery research. Due to the difficulty in accurately predicting aqueous solubility in silico, high throughput experimental determination of aqueous solubility is in great demand. This study evaluates a method using a multi-wavelength UV plate reader and disposable 96-well UV plates for fast solubility determination. It was demonstrated that this method has the sensitivity and reproducibility to effectively determine solubility as low as 1 μM. Excellent correlation (R > 0.97) was observed between the solubility determined using the UV reader method and the HPLC method over the range of 1-1000 μM for a diverse set of pharmaceutical compounds. In addition to excellent sensitivity and reproducibility, the UV plate reader method also offers the flexibility of being able to determine thermodynamic solubility in the presence or absence of dimethyl sulfoxide, which is a solvent widely used for combinatorial compounds during high throughput screening.