Publisher Summary Soluble glycerol-3-phosphate dehydrogenase in Drosophila melanogaster represents a family of three distinct isozymes that are designated as glyceraldehyde 3-phosphate dehydrogenase (GPDH)-1, -2, and -3 in order of their decreasing mobility toward the anode during starch gel electrophoresis. The multiple forms of glycerol-3-phosphate dehydrogenase in Drosophila are the product of the same structural gene mapped to the left arm of chromosome II and arise by an epigenetic mechanism. This chapter describes the assay method and properties of sn -glycerol-3-phosphate dehydrogenase isolated from Drosophila melanogaster . Glyccrol-3-phosphatc dehydrogenase activity is determined spectrophotomctrically at 340 nm using glycerol 3-phosphate or dihydroxyacetone phosphate as a substrate and measuring the rate of nicotinamide adenine dinucleotide kinase (NAD + ) reduction or nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation. The assay utilizing glycerol 3-phosphate is convenient and is used routinely. The steps involved in the purification procedure of GPDH-1 and GPDH-3 are (1) ammonium sulfate fractionation, (2) diethylaminoethyl (DEAE)-column chromatography, and (3) adenosine tripjosphate (ATP)-Sepharose 4B chromatography.