An analytical method for the detection and quantification of anthracene from interstitial fluid samples was developed by using Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry (APCI-MS/MS). The anthracene samples were obtained using intradermal microdialysis to assess dermal absorption of this polycyclic aromatic hydrocarbon (PAH). The experimental considerations were evaluated based on the chemical properties of this PAH and the detection limits of the instrumentation. The addition of an isotopically labeled internal standard allows the reduction of ionization suppression due to instrumental fluctuation and run-to-run deviation. The dermal extraction samples were prepared considering the proceeding conditions for measurement enhancement. Several variables for method validation including coefficient of determination (R2 = 0.993 ± 0.003), percent error (% error = 0 ± 2%), coefficient of variation (% CV = 5 ± 1%), lowest limit of detection (LOD = 39 ± 3 ng mL-1), and lowest limit of quantification (LLOQ = 129 ± 10 ng mL-1) were obtained from the inter- and intra-day measurements for the calibration and the quality control samples. Posterior to this, actual dermal interstitial fluid samples were measured, and it was observed they fitted into the ranges defined from the method development with good accuracy and precision. It was observed that the introduction of an internal standard while performing APCI-MS/MS allows an accurate and precise measurement of the concentration of anthracene to be obtained from dermal extraction samples. This method could be further used for complex mixtures to enhance our understanding of hazardous exposure of PAH on firefighter gear.