The present study provides a comprehensive assessment of the quantitative analysis by high-performance liquid chromatography coupled with dual orthogonal electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) of pinene markers, biomass-burning related phenols, and other relevant carboxylic acids in atmospheric aerosol samples. Significant insights into the quantitative determination are offered on the basis of systematic experiments targeting the optimization of chromatographic separation, ionization source, and mass spectrometer performance. After testing three analytical columns, the best separation of the compounds of interest was achieved on a Poroshell 120 ECC18 column (4.6 × 50 mm, 2.7 µm) thermostated at 35 °C, operating in gradient elution mode with 0.1% acetic acid in water and acetonitrile at a 0.8 mL min−1 flow rate. Optimal operational conditions for the ESI-TOF-MS instrument were identified as a 350 °C drying gas temperature, 13 L min−1 drying gas flow rate, 60 psig nebulizer pressure, 3000 V for the ion transfer capillary, 60 V for the skimmer, and 150 V for the fragmentor. Additionally, the matrix effect on the ESI efficiency and the spike recovery factors of the compounds were tested. Method quantification limits can go as low as in the 0.88–48.0 μg L − 1 (3.67–200 pg m − 3, at 120 m3 of sampled air) range. The developed method was shown to be reliable for the quantification of the targeted compounds in real atmospheric aerosol samples. The accuracy in the molecular mass determination of less than 5 ppm and the acquisition in the full scan mode were shown to bring additional insights into the organic constituents in atmospheric aerosols.