An accurate and precise Multiple Headspace Solid-Phase Microextraction (MHS-SPME) method was developed and validated for quantifying the volatile composition of White and Tawny Port wines. SPME extraction conditions were optimised using a four-factor three-level Box-Behnken design with three blocks and two replications. Optimal extraction conditions were similar for both Port wines. The method showed good linearity (0.001-50mg/L), precision (<5%), and detection limits (<1μg/L), well below the olfactory detection threshold. Recoveries higher than 95% were obtained. Twenty-three aroma compounds were quantified in Tawny and, for the first time, in White Port wines, including five acids, fourteen esters, the most abundant class, and four norisoprenoids, whose levels apparently increased with age. White Port wines had a lower abundance of aroma compounds. Results show that this MHS-SPME method is suitable for analysing volatile composition of White and Tawny Port wines, with reduced costs, manipulation time and eliminating matrix effects.