Simultaneous determination of D- and L-lactic acids was achieved using an apparatus containing two enzyme reactors and an octadecylsilica column placed between the two reactors in tandem in a single flow line. The enzymes used for the D-lactic acid assay were D-lactate dehydrogenase and glutamic-pyruvic transaminase, and for the L-lactic acid assay, L-lactate dehydrogenase and glutamic-pyruvic transaminase. The optimal concentrations of L-glutamic acid and NAD+in the carrier were determined. Of the buffers examined for use as a carrier medium, Tris buffer was found to be the most favorable. Under these optimal conditions, NADH due to D-lactic acid and that due to L-lactic acid could be observed separately in this apparatus. The calibration curve for D-lactic acid was linear in the range of 20 – 1000 μM, and that for L-lactic acid was 2 – 250 μM. This method was applied to analyze D- and L-lactic acids in several beverages that contained lactic acid bacteria. The D- and L-lactic acid content determined by the present method agreed with that determined by a commercially available test-kit method.