Cancer is a pathology characterized by presenting a high proliferation, heterogeneity, and supervising of abnormal cells. In environments such as hypoxia and irradiation, the tumor cells have changes in the expression of genetics and metabolic that allow them to adapt to the demands of the tumor microenvironment. Through procedures such as PET, we can use glucose uptake as a method to predict the response of tumors, taking advantage of the high glycolytic flow of tumor cells. This work aimed to evaluate the effect of hypoxia and irradiation on glucose uptake in tumor lines of breast and colon cancer using a glucose analog. Here we describe a protocol that uses the fluorescent glucose analog (2-NBDG) in cell lines of MCF-7, HT-29, and EA.hy926 under different experimental treatments, followed by processing the fluorescence images. The results indicate a decrease in uptake glucose in hypoxia and reoxygenation treatment in EA.hy926, MCF-7, and HT-29 cell lines. Whereas, in the irradiation treatment, a significant increase was observed only in the HT-29 tumor line concerning the control. It's suggested that the reoxygenation period had a positive effect on MCF-7 and EA.hy926 cells, while in the HT-29 tumor cells wasn't established any relation and on the contrary, it's an indication of possible radioresistance.