Metabolic reprograming is an established hallmark of cancer cells. Pancreatic cancer cells, by virtue of the underlying oncogenic drivers, demonstrate metabolic reprograming to sustain growth, invasiveness, and therapy resistance. The increased demands of the growing tumor cells alter the metabolic and signaling pathways to meet the growing nutrient requirements. Investigating the metabolic vulnerabilities of tumor cells can help in developing effective therapeutics to target pancreatic cancer. In this chapter, we explain in detail the methods to evaluate the metabolic changes occurring in the tumor. This includes the glucose/glutamine uptake assays and the measurement of reactive oxygen species, extracellular acidification rate, and oxygen consumption rate in the tumor cells. All these physiological assays help in understanding the metabolic nature of the tumor.