Human acetyl-coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium-adenosine triphosphate complex to magnesium-adenosine diphosphate complex. A simple off-column capillary electrophoresis assay for human acetyl-coenzyme A carboxylase 2 was developed based on the separation of magnesium-adenosine triphosphate complex, magnesium-adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl-coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2, 2.5 mM KHCO3, and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme-catalyzed reaction (off-column). Inhibition of human acetyl-coenzyme A carboxylase 2 by CP-640186, a known inhibitor, was detected using the capillary electrophoresis assay.