Bio-ethanol is produced from biomass or biodegradable waste and is used as a blending component in commercial gasoline fuel. Bio-ethanol can be contaminated with chloride and sulfate ions which can cause various unwanted side effects. European norm, EN 15376 defines the maximum permitted level of ions present and by that maximum chloride and sulfate content should not exceed 6.0 mg.kg(-1) and 4.0 mg.kg(-1), respectively. The optimization and validation of ion chromatography method for the quantitative determination of chloride and sulfate ions in bio-ethanol by direct injection ion chromatography using anion-exchange stationary phase, carbonate/hydrogen carbonate mobile phase and conductivity detection has been described. The method is optimized regarding mobile phase composition and flow rate as well as the length of the chromatographic column. Validation of the method has been performed in order to confirm its applicability. The developed method is applied for the determination of chloride and sulfate ions in real bio-ethanol samples used for inter-laboratory comparative testing. The obtained results are compared with the official tests results and z-score has been determined. For chloride determination higher z-scores is obtained (from 1.4 to >3) than for the determination of sulfate (from -0.9 to 0.1).