© 2022 Elsevier B.V.Temozolomide (TMZ) is the first-line chemotherapeutic agent for the treatment of newly diagnosed glioblastoma (GBM). However, chemoresistance, hydrolytic instability, and insufficient drug accumulation are major challenges limiting the effectiveness of TMZ chemotherapy. Although various nanocarriers have been offered to overcome these limitations, there are some discrepancies regarding the TMZ encapsulation efficiency (EE%). In this study, by examining the behavior of TMZ in different solvents, we aimed to confirm the loading efficiency of TMZ in the nanoparticles (NPs) and investigate the therapeutic efficacy of TMZ-loaded NPs in human GBM cell line (T98G). For this purpose, firstly, we investigated the stability of TMZ and its degradation product (AIC) to evaluate whether TMZ degradation affects the measured EE% from the supernatant (indirect method) or the NPs (direct method). Subsequently, we tried to load TMZ into magnetic poly (lactic-co-glycolic acid)-polyethylene glycol (PLGA-b-PEG) nanoparticles (TMZ-m-PNPs) by modifying the synthesis parameters in a controlled manner. It was concluded that the indirect method might cause misleading results due to the high hydrolytic instability of TMZ during the NPs synthesis or washing steps. Although many modifications have been performed in the synthesis method, the EE% of TMZ ranged between 1% and 7%. In cell culture, IC50 values of free-TMZ were found for human endothelial cells (HUVEC, ∼500 μM) and human glioblastoma cells (T98G, ∼350 μM) at the 72nd h. Furthermore, cell viability was quantified using MTT, live/dead cell viability, and Annexin V-FITC/PI assays of TMZ-m-PNPs, but cytotoxicity of NPs was not dose-dependent in T98G cells. Overall, this study offers two major new insights; first, the EE% of TMZ in NPs should be determined by the direct method; second, to increase the therapeutic efficacy of TMZ-loaded NPs on GBM cells, it may be recommended not to use the PLGA-based nanocarrier system that may contribute to poor therapeutic response.