The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100 mM NaOH+40 mM NaNO(3). Orotic acid was determined at the concentration ranges of 0.2-30 microM (r=0.9997) with an absolute detection limit of 80 pg (10 microL injected). The levels of OrA in cows' milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheep's milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.