Excessive excretion of orotic acid in urine arises as a result of some inborn metabolic diseases such as urea cycle disorders and pyrimidine synthesis disorders. Therefore, rapid determination of orotic acid in the neonatal period is vital. Existing methods for measuring orotic acid levels in urine are both time consuming and expensive. Consequently, rapid definition of the orotic acid level in human urine is important. In this current study a novel, simple, cost effective and rapid (fast) capillary electrophoretic analysis method was developed for quantitative measurement of orotic acid content in urine samples. Determination of orotic acid by employing capillary electrophoresis coupled with contactless conductivity detection was achieved by using, 20 mM 4-Morpholineethanesulfonic acid (MES), 10 mM Histidine, 0.1 mM Cetyltrimethylammonium Bromide (CTAB) at pH of 6.5 as optimum electrolyte solution. Orotic acid was well separated from the sample constituents within a short analysis time less than 3.5 minutes. The precisions for the migration times and corrected peak areas were calculated as 1.25 and 4.31 RSD% respectively. Limit of detection and limit of quantification of the proposed method were also calculated as 0.733 mg / L and 2.443 mg / L respectively. The developed sensitive and facile capillary electrophoresis method was validated and applicability of the method was successfully demonstrated by assessment of orotic acid content in urine.