The benzene metabolite trans,trans-muconic acid (tt-MA) is widely used as a biological indicator of exposure to this xenobiotic. An analytical method was developed and validated for the determination of urinary tt-MA using solid phase extraction onto a Strata SAX cartridge. The analysis was performed by HPLC with an Aminex HPX-87H ion exclusion column and a UV diode array detector. The sample preparation conditions were optimized using a 2⁴⁻¹ factorial design. Comparison of the slopes of standard analytical curves prepared in aqueous solution and urine showed that the biological matrix suppressed the tt-MA signal by around 50%, so the analytical curves were prepared with tt-MA standard at low concentrations in pooled urine. The analytical curves in the range of 5–500 μg·L⁻¹ showed determination coefficients values (R²) > 0.99 for tt-MA standard in water and R² > 0.98 for tt-MA standards in pooled urine. The coefficients of variation obtained using seven replicates were lower than 3.6%, and recoveries of tt-MA from solutions containing 5, 25, 50 and 100 μg·L⁻¹ of the analyte were in the range 85%–90%, demonstrating the satisfactory precision and accuracy of the method. The limits of detection and quantification were 0.11 and 0.36 µg ·L⁻¹, respectively. The benefits of this new method developed is the possibility of complete chromatographic peak separation for the determination of tt-MA at baseline, without matrix components’ interference as normally found in the C18 column. This is the first time that this chromatographic column has been used for the analysis of tt-MA in urine.