During G6PD deficiency, the red cells are unable to regenerate reduced Nicotine adenine dinucleotide phosphate (NADPH), a reaction that is normally catalyzed by the G6PD enzyme. Since the X chromosome carries the gene for G6PD enzyme, this deficiency mostly affects the males. The two major conditions associated with G6PD deficiency are hemolytic anaemias and neonatal jaundice, which may result in neurological complications and death. Screening and detection of G6PD deficiency helps in reducing such episodes, through appropriate selection of treatment, patient counselling and abstinence from disease precipitating drugs such as anti malarials and other agents.

G6PDH in the RBC's is released by a lysing agent present in the reagent. The G6PDH released catalyzes the oxidation of Glucose 6 phosphate with the reduction of NADP to NADPH. The rate of reduction of NADH to NADPH is measured as an increase in absorbance, which is proportional to the G6PDH activity in the sample.