his study represents, detailly, the validated method for the extraction and quantification of widespread phthalic acid esters (PAEs) bis(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP) and di-n-octyl phthalate (DnOP) from solitary ascidians collected from a marine environment. The extraction was based on a pressurized liquid extraction method, using n-hexane as the solvent to extract the target PAEs from dry biological tissues, and was performed in an accelerated solvent extraction instrument. The average recovery of 89.2% was obtained from samples subjected to a pressure of ~1500 psi and 120°C in two 10-min cycles. GC-MS was used for quantification, conducted in single-ion monitoring mode. Following careful and rigorous cleanup procedures to prevent cross-contamination from laboratory glassware, PAE standards showed signals with good specificity. The obtained limits of detection were 130, 122 and 89 ng/g for DEHP, DBP and DnOP, respectively. Accordingly, the calculated limits of quantification were 394, 370 and 270 ng/g for DEHP, DBP and DnOP, respectively. The obtained linearity ranged from 5.4 to 269 ng/ml (equivalent to 135 - 6725 ng/g dry weight), with R2 ≥ 0.998. Concentrations in the range of 200 to 9000 and 400 to 5000 ng/g sample dry weight, for DEHP and DBP, respectively, were obtained from the ascidians. No DnOP was detected in any of the samples. These results indicate that the method presented in this study is applicable for detection of low and trace concentrations of the target PAEs in samples collected from a marine organism, which can serve as a bioindicator of plastic contamination.