This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman′s reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.