A host of critical metabolic and biosynthetic processes are mediated hepatocytes, the dominant cell type found in the liver. Despite decades of literature documenting the isolation and characterization of isolated hepatocytes, the overwhelming majority of investigations center on either the metabolic or xenobiotic arm of hepatic function. While regulation of metabolic substrates and detoxification of xenobiotic compounds are independently crucial, these functions are invariably interrelated. For most investigations, metabolic functions are arguably of greater interest due to their physiological relevance; even in drug metabolism studies, the overarching questions often center upon whether a compound affects key biosynthetic processes (i.e. glucose synthesis). Furthermore, extensive evidence suggests that metabolic functions (i.e. synthesis of glycogen and oxidation of fatty acids) are more sensitive markers for hepatocyte integrity than xenobiotic processes (i.e. induction of cytochrome P450-1-A1 activity). However, isolation of high-quality hepatocytes suitable for metabolic investigations is challenging; this issue has been compounded by a lack of standard reference ranges for comparison purposes. In this dissertation, a means for isolation of fully competent primary mouse hepatocytes is detailed, along with an array of metabolic and xenobiotic assays designed to validate their performance. Key biosynthetic rates are provided relative to published literature, and proof of concept is provided by application of these tools towards fundamental questions concerning the specific versus nonspecific impact of dioxin-like polychlorinated biphenyls on hepatic glucose metabolism. Together, these investigations provide the framework for future mechanistic studies that integrate multiple aspects of liver function in a reproducible, high-throughput manner.