A new etherified starch, δ-hydroxybutyl starch (δ-HBS), was prepared by utilising 4-chlorobutan-1-ol as the etherifying reagent. The method of Zeisel gas chromatography for the determination of the molar substitution was described. This technique offers a simple and rapid method for quantitative analysis with reproducible results. Meanwhile, the mechanism of the Zeisel reaction was also investigated.