Background: A micellar electrokinetic chromatographic (MEKC)/ indirect UV detection method with hydrodynamic and electrokinetic injection has been developed for the determination of pregabalin in the serum samples. Methods: Separation of the drug was achieved on Agilent capillary electrophorese in less than 5 min using a 50 cm × 75 μm i.d. uncoated fused-silica capillary and a background electrolyte (BGE) consisting of 5-aminosalicylic acid (5-ASA, 10 mmol L-1), cetyl trimethylammonium bromide (CTAB, 1 mmol L-1) and tri-sodium citrate (4% w/v). The influence of various parameters on the separation such as separation voltage, injection time, cassette temperature, pH of BGE and organic modifier was investigated. Results: Method validation shown good linearity (R2> 0.999) in the range of 1.5-100 µg mL-1 of pregabalin. A limit of detection (LOD) of 0.8 μg mL-1 and a limit of quantitation (LOQ) of 2.6 μg mL-1 were reported for pregabalin. Conclusion: The proposed method was found to be suitable and accurate for the determination of pregabalin in serum samples.