A sensitive fluorometric assay for the determination of dihydroxyacetone phosphate (DHAP) is reported here. DHAP is reduced to l-glycerol-3-phosphate with NADH-dependent α-glycerophosphate dehydrogenase. DHAP recycling is provided by oxidation reaction catalysed by α-glycerophosphate oxidase to release hydrogen peroxide. The reaction of hydrogen peroxide with Amplex® Red reagent under horseradish peroxidase catalysis leads to the fluorescent product resorufin. The limit of detection of DHAP is estimated at 1 pmol which is roughly 2250 fold more sensitive than the usual DHAP assay based on the detection of NADH by spectrophotometry. This assay is ready-to-use for automated medium-throughput screening.