Oxidative damage due to low levels of glutathione (GSH) is one of the main causes of cataract formation. It has been reported that 2-oxothiazolidine-4-carboxylic acid (OTZ), a cysteine prodrug, can increase the cellular level of GSH. Currently, there is no analytical method to separate and quantify OTZ from aqueous humour samples for cataract research. The present study aims to develop and validate a hydrophilic interaction liquid chromatography (HILIC) method for the quantification of OTZ in simulated aqueous humour (SAH). The developed method was validated according to FDA guidelines. Accuracy, precision, selectivity, sensitivity, linearity, lower limit of quantification (LLOQ), lower limit of detection (LLOD) and stability were the parameters assessed in the method validation. The developed method was found to be accurate and precise with LLOQ and LLOD of 200 and 100 ng/mL, respectively; method selectivity was confirmed by the absence of any matrix interference with the analyte peak. The constructed calibration curve was linear in the range of 0.2–10 μg/mL, with a regression coefficient of 0.999. In addition, the OTZ was found to be stable in SAH after three freeze/thaw cycles. Chitosan nanoparticles loaded with OTZ were formulated by the ionic gelation method. The nanoparticles were found to be uniform in shape and well dispersed with average size of 153 nm. The in vitro release of OTZ from the nanoparticles was quantified using the developed analytical method over 96 h. Permeation of OTZ through excised bovine cornea was measured using HILIC. The lag time and the flux were 0.2 h and 3.05 μg/cm2 h, respectively.