This study describes the development of a new ITO-based electrode used for the electrochemical determination of N-acetyl-L-cysteine (NAC). The electrode system relies on in situ grown CuO nanostructures over the ITO substrate, which provide a relatively greater contact and enables effective electron facilitation during the electrocatalytic oxidation of NAC in an aqueous medium. In situ growth of the CuO nanostructures was achieved using a simple hydrothermal process with the application of succinic acid as an effective growth template. The grown layer of CuO possessed a dense population of highly ordered nanostructures with a high degree of structural uniformity. The study evaluates the potential of the newly developed ITO-based electrode against the bare GCE and an electrode modified via the direct deposition of the same CuO nanostructures synthesised under similar hydrothermal conditions. The electrochemical oxidation of NAC over the newly developed electrode demonstrated low-over potential value and good working linearity in the range from 0.01 to 0.28 mu M. The electrode system was found to be sensitive up to 1.2 x 10(-3) mu M (S/N = 3) with charge transfer co-efficient (alpha) and diffusion co-efficient (D) values of 0.65 and 1.62 x 10(-2) cm(2) s(-1), respectively. Moreover, the developed electrode system demonstrated excellent working capability when utilised for the determination of NAC from a pharmaceutical formulation obtained from a local pharmacy.