The method for the determination of biotin by high performance liquid chromatography (HPLC) coupled with coulometric detector is presented here. Chromatographic and detection conditions were tested. A LiChrospher 60RP-select B column (250 mm x 4 mm; 5 microm) and the mobile phase containing 0.24 mol/L aqueous solution of acetic acid and acetonitrile in the ratio 85:15 (v/v) were found as the most suitable. The flow rate was 1 mL/min and the injected volume of the sample was 20 microL. The hydrodynamic voltammogram of biotin was measured and according to obtained data the detection parameters were set--channel I 600 mV, channel II 900 mV, sensitivity 1 microA. The developed method has been validated. The calibration curve is linear in the range 15-3600 ng/mL, correlation coefficient is 0.9998, limits of detection and quantification are 5 and 15 ng/mL, respectively. Recovery of the spiked samples was 98.67% with R.S.D. 0.255% on average. The developed method has been successfully applied for determination of biotin in pharmaceutical preparations.