The purpose of this work is to introduce a rapid and practical method for quantitatively analyzing total acid number in biodiesel by a reaction headspace gas chromatography. In this method, the carbon dioxide formed by the reaction of acidic species (i.e., fatty acids) in biodiesel and sodium bicarbonate can be measured by GC. The results showed that the complete reaction can be achieved in 25 min at 75 degrees C. The relative standard deviation of this method in total acid number determination was within 2.93%, the relative differences has been determined by comparing the total acid number obtained from this new analytical technique with the data from the reference technique (i.e., potentiometric titration), which are less than 8.05%. The reaction HS-GC technique is automated, efficient and can be a reliable tool for quantifying the total acid number in biodiesel and related research.