This essay presents a reliable, effective and easy procedure for measuring glutathione peroxidase activity (Gpx). The enzyme samples were incubated with phosphate buffer, which included appropriate concentrations of glutathione and peroxide as substrates, to determine the Gpx activity. After a sufficient incubation time, the CUPRAC reagent (Cu(Nc) 2 2+ ) was added to stop the enzyme’s reaction. The unreacted substrates act to reduce Cu(II)-neocuproine complex (Cu(Nc) 2 2+ ) to strongly coloured Cu(I)-neocuproine complex (Cu(Nc) 2 + ) that was measured spectrophotometrically at 450 nm (CUPRAC method). The glutathione peroxidase activity was linked to a decrease in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc) 2 + ). The procedure uses the Box–Behnken design (BBD) to optimise the formation of the Cu(I)-neocuproine complex (Cu(Nc) 2 + ). The response surface methodology (RSM) is used to determine the accuracy of the method. This new protocol was confirmed by applying a Bland–Altman plot analysis of Gpx activity in matched samples using the Gpx-DTNB assay. The correlation coefficient between the two protocols was 0.9967. This means that the new protocol was very accurate and on par with the comparison method.