A procedure to assess the bioavailability of persistent organic pollutants (POPs) from soil samples has been developed. The procedure is based on the use of simulated in vitro gastrointestinal extraction to remove POPs from soil matrices. The level of recovery, using this approach, is assessed following liquid-liquid extraction (LLE) and analysis by gas chromatography-mass selective detection (GC-MSD). The remaining soil residue is then extracted using pressurised fluid extraction (PFE) followed by GC-MSD analysis to assess the residual fraction. The residual fraction is monitored to determine the unavailable fraction i.e. not available for absorption in the gastrointestinal tract of humans. The procedure was applied to four soil samples i.e. an aged, spiked soil and three certified reference materials (CRMs) contaminated with POPs. Recoveries of pesticides (lindane, endosulfan I, endrin, DDE, DDD and endosulfan II), phenols (cresol, TCP and PCP), and base neutral compounds (hexachloroethane, acenaphthene, dibenzofuran, fluorene and hexachlorobenzene) from aged, spiked soil following extraction with gastric fluid ranged from 0.8 to 8.3% while following intestinal extraction ranged from 5.5 to 13.5%, irrespective of POP. Recoveries of pesticides (lindane, endosulfan I, endrin, DDE, DDD and endosulfan II) from CRM 805-050 following extraction with gastric fluid were below the limit of detection while following intestinal extraction ranged from 5.3 to 12.8%. Recoveries of phenols (cresol, TCP and PCP) from CRM 401-225 following extraction with gastric fluid ranged from 1.6 to 2.0% while following intestinal extraction ranged from 4.1 to 5.4%. Recoveries of base neutral acid analytes (hexachloroethane, acenaphthene, dibenzofuran, fluorene and hexachlorobenzene) from CRM 107-100 following extraction with gastric fluid ranged from 1.4 to 4.0% while following intestinal extraction ranged from 6.6 to 12.7%. It has been found that the majority of POPs present i.e. >75%, would be excreted if consumed and not be absorbed in the gastrointestinal tract of humans.