Toxicology research into the global public health burden of fine particulate matter (PM) exposures frequently requires extraction of PM from filters. A standardized method for these extractions does not exist, leading to inaccurate inter-laboratory comparisons. It is largely unknown how different filter extraction methods might impact the composition and bioactivity of the resulting samples. We characterized the variation in these metrics by using equal portions of a single PM filter, with each portion undergoing a different extraction method. Significant differences were observed between extraction methods for concentrations of elements and polycyclic aromatic hydrocarbons (PAHs) of the PM tested following its preparation for biological response studies. Importantly, the chemical profiles differed from those observed when using standard protocols for chemical characterization of the ambient sample, demonstrating that extraction can alter both chemical concentrations and composition profiles of the extracts. The impact of these chemical differences on sensitive endpoints of zebrafish development was investigated. Significant differences in the percent incidence and timing of mortality were associated with PM extraction method. This research highlights the importance of and rationale for considering extraction method when making inter-laboratory comparisons of PM toxicology research.