<p>Urban air pollution is heavily influenced by particulate matter, which often contains a complex mixture of organic molecular markers indicative of various sources. This study presents a comprehensive, validated analytical protocol for the simultaneous quantification of 136 target compounds across five major families of organic markers, categorized into five diverse families: 64 phenols, 22 carboxylic acids, 16 polycyclic aromatic hydrocarbons (PAHs), 14 saccharides, and 20 tire wear particle (TWP) chemicals. The protocol employs a dual-step accelerated solvent extraction process to effectively recover both polar and non-polar analytes: an initial extraction with acetonitrile targets phenols, carboxylic acids, PAHs, and TWP chemicals, followed by dichloromethane:methanol (1:1 v/v) for saccharides. Extracts are analyzed using chromatographic techniques tailored for each class: gas chromatography-mass spectrometry (GC-MS) for phenols and carboxylic acids, ion trap GC-MS for saccharides, gas chromatography-tandem mass spectrometry for PAHs, and high-performance liquid chromatography-tandem mass spectrometry for TWP chemicals, confirming good linearity (<i>r</i>² &gt; 0.99), acceptable recovery rates (median of 80%), low detection limits, and consistent intra- and inter-day precision (RSD &lt; 20%). The method was applied to 14 PM₁₀ samples collected from a semi-urban area highly influenced by traffic, industrial, and residential sources. The quantified concentrations in all compound classes exceeded the method’s limits of quantification and limits of detection, confirming its suitability for trace-level detection in complex urban environments.</p> Graphical Abstract <p></p>