Hypoxic microenvironment found in pancreatic ductal adenocarcinoma and other solid tumors is central to physiological and metabolic alterations of immune cells that significantly impact tumor growth dynamics. Hypoxic adaptations in the immune cells are primarily mediated by the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which regulates cellular metabolism by modulating glycolysis and other interconnected metabolic pathways. HIF-1α plays distinct roles in M1 and M2 macrophage polarization, which, in turn, regulates tumor cell immune escape and growth. In this chapter, we describe a real-time PCR-based assay to monitor the transcript levels of Arg1 and Nos2 to assess the status of tumor-induced macrophage polarization under hypoxic conditions. This method can be effectively utilized to delineate the genes critical for M1/M2 polarization in the hypoxic tumor microenvironment and would provide opportunities to develop immunomodulating therapies to regulate the tumor growth, progression, and metastatic dissemination.