A large number of epidemiological studies have demonstrated that increased morbidity and mortality are associated with increased atmospheric particulate matter (PM) concentrations. Although many of these studies have used PM mass as an exposure metric, there is increasing epidemiological and toxicological evidence that exposure to specific components of particulate matter are the drivers of adverse health outcomes. To better understand the role of different components of PM and biological pathways of disease associated with PM exposures, laboratory tools have been developed to elucidate the chemistry of real world PM samples that control biological activity observed in in-vitro assays. These tools include detailed chemical speciation of real world particulate matter samples and chemical fractionation these samples that are suitable for in-vitro assays. Complementing these tools, an in-vitro macrophage assay has been developed that has been shown to be associated with human biomarkers of inflammation. Current research will be presented that applies these tools to the macrophage assay used to measure reactive oxygen species (ROS) and TNF-α production, which allows the macrophage assay to provide a direct link PM chemistry and human health studies. The macrophage assay and chemical speciation tools have been applied to particulate matter samples collected in Los Angeles, Denver and Lahore, Pakistan, to elucidate the impact of chemistry, sources and atmospheric processing on biological activity of PM. The components of PM that drive different endpoints of the macrophage assay will be discussed along with the implication for human health effects.