Polychlorinated biphenyls (PCBs) are a class of synthetic, aromatic, organic chemicals that were produced as complex commercial mixtures. Although the production and use of PCBs is now banned due their lipophilic, bioaccumulative, persistent, and toxic properties, they are still one of the most ubiquitous pollutants in the environment. Currently, several validated methods for the quantitation of PCBs from biological matrices exist; however, they often employ the use of expensive, hazardous, or tedious extraction methods. Specifically, previous methods rely heavily on the use of large solvent volumes, multiple extractions, evaporation techniques, extensive clean-up procedures, and potential derivatization of analytes; thereby resulting in time consuming, cost intensive, low throughput sample procedures. The chapters herein present a unique, simple, cost-efficient, single-step cleanup method for limited sample amounts that allows for the sensitive and selective quantitation of both low and highly chlorinated PCB congeners via a novel extraction method for brain and liver using the Phenomenex PHREE 96-well plate and gas chromatograph tandem-mass spectrometry system. Furthermore, this analytical method holds important implications for the detection of low level exposure of persistent organic pollutants in complex biological matrices. Thus, this research lays the foundation for the future evaluation of physiologically relevant PCB congeners as neurodevelopmental toxicants.