<p>A micellar electrokinetic chromatography (MEKC) methodology was optimized for the indirect determination of glycerol based on formaldehyde formation and the use of 3-methyl-2-benzothiazolinone hydrazone (MBTH) as a derivatizing agent. A Taguchi experimental design matrix L₉(3³) was used to evaluate the critical variables and maximize the separation efficiency. Under optimum conditions of [SDS] 60.0 mM, [Na₂B₄O₇] 25.0 mM, and a pH value of 9.0, the methodology provides a linear interval range (0.18–0.91% w/w), a limit of detection (0.06% w/w), with satisfactory results in terms of intra-day and inter-day precision %RSD &lt; 6.0% in all cases. The proposed methodology was applied to real biodiesel samples, and the results were compared with the official standard methodology in the determination of glycerol content (%w/w). The analysis of eight samples yielded results ranging from 0.12 to 0.20% w/w, and no differences were observed. The proposed methodology was applicable to quantifying glycerol at the concentration levels established by international norms.</p> Graphical abstract <p></p>