The assay involves the oxidation of glucose-6 phosphate (G-6-P) substrate to 6-phosphogluconate (6-PG), by the G6PD present in the  blood spot sample and the concomitant reduction of NADP to NADPH. The blood spot is  allowed to react with the substrate reagent which consists of G-6-P and NADP, for 30  minutes at ambient temperature. Copper sulfate is added to slow down the reaction. The fluorescence is measured using an excitation wavelength of 355 nm and an emission wavelength of 460 nm.