A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.