The application of substrate cycling for alpha-ketoglutarate (AKG) through enzymatic amplification has been demonstrated in conjunction with capillary electrophoresis. AKG is determined using an off-line assay, which involves the coupled enzymatic reactions catalyzed by glutamate dehydrogenase and glutamic oxalacetic transaminase. Varying concentrations of the substrate and set concentrations of cofactors and enzyme are incubated together outside the CE instrument. The accumulated product, nicotinamide adenine dinucleotide, reduced form (NADH), was detected at 340 nm using CE. Amplification of a factor of 10 over just the reversed glutamate dehydrogenase reactions permitted an AKG detection limit of 10 microM. Throughput for each assay is about 5 min (including wash and equilibrium steps). After simple dilution as the only sample pretreatment, spiked AKG serum samples were determined with an average recovery of 96% with a 3% RSD.