A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD(+)/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP(+) and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD(+) and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.