Hypoxic conditions in the pancreatic tumor microenvironment lead to the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which acts as the master regulator of cancer cell metabolism. HIF-1α-mediated metabolic reprogramming results in large-scale metabolite perturbations. Characterization of the metabolic intermediates and the corresponding metabolic pathways altered by HIF-1α would facilitate the identification of therapeutic targets for hypoxic microenvironments prevalent in pancreatic ductal adenocarcinoma and other solid tumors. Targeted metabolomic approaches are versatile in quantifying multiple metabolite levels in a single platform and, thus, enable the characterization of multiple metabolite alterations regulated by HIF-1α. In this chapter, we describe a detailed metabolomic approach for characterizing the hypoxia-induced metabolomic alterations using pancreatic cancer cell lines cultured in normoxic and hypoxic conditions. We elaborate the methodology of cell culture, hypoxic exposure, metabolite extraction, and relative quantification of polar metabolites from normoxia- and hypoxia-exposed cell extracts, using a liquid chromatography-coupled tandem mass spectrometry approach. Herein, using our metabolomic data, we also present the methods for metabolomic data representation.