Petroleum substances are a group of complex substances with constituents having a wide range of physicochemical properties and abundances. Due to the complexities and difficulties in obtaining detailed compositional analysis of petroleum susbtances, it is not practical to evaluate the toxicity based on a hazard assessment for each constituent. Bulk exposure metrics, such as total polycylclic aromatic hydrocarbons (TPAH) or total petroleum hydrocarbons (TPH) have been used as hazard assessment metrics to screen the toxicity of petroleum substances. However, these methods are not comparable across petroleum susbtances and do not provide a consistent basis for establishing effect levels partially due to biased measurements due to different constituent solubilities. Passive sampling is a flexible platform for measuring bioavailable concentrations of environmental contaminants, including complex substances. This presentation will discuss the development of simple passive sampling analytical techniques, based on solid phase microextraction (SPME) to measure the bioavailability of complex petroleum substances. The advantage of this approach is using existing SPME and GC-FID technologies in combination to develop measurements of bioavailable material, a so-called “biomimetic extraction (BE).” This method for measuring bioavailable fractions for complex substances was validated against high quality toxicity data. The resulting fiber-based effect thresholds are compared to conventional, tissue threshold-based methods. The BE method has been applied to a wide variety of matrices including refinery effluents, oil sand process water, and contaminated sediments. Results of this method are used to support management of sediment and wastewater and also to design toxicity testing programs, and are used to support toxicity identification evaluation of complex effluents.