Nanomedicine has been, to a certain degree, a success story in the development of superior anticancer therapies. However, there are tumors that remain a huge challenge for nanoformulations, for instance, brain tumors such as glioblastoma, the most common and aggressive brain tumor. To utilize the fact that such tumors are characterized by an acidic extracellular environment, we selected pH-responsive liposomes as a potential drug delivery system for superior delivery to GBM. Liposomes comprising PEGylated lipid of two chain lengths with encapsulated fluorescent marker calcein were characterized and challenged against non-PEGylated vesicles. The in vitro calcein release from three liposomal formulations (<200 nm), namely non-PEGylated (pH-Lip) and PEGylated, pH-Lip–PEG750, and pH-Lip–PEG2000, was followed at three pH conditions to prove the pH-responsiveness. The intracellular delivery of a liposomally encapsulated marker was determined in GL261 glioblastoma cell lines in vitro using both flow cytometry and confocal microscopy. The inclusion of PEG2000 within liposomal formulation resulted in reduced in vitro pH-responsiveness compared to pH-Lip and pH-Lip750. All three pH-responsive liposomal formulations improved intracellular uptake in GL261 cells compared to non-pH-responsive liposomes, with negligible differences regarding PEG length. The proposed formulations should be further evaluated in glioblastoma models.