Glioblastoma is the most common primary malignant brain tumor. It is aimed to elucidate the role of ROCK in the regulation of PI3K signaling inhibited by PTEN and to evaluate the effects of ROCK inhibitors Thiazovivin and GSK 429286 on glioblastoma as a new therapeutic strategy. U87 MG and L929 cell lines were used to research the cytotoxic effect of ROCK inhibitors Thiazovivin and GSK 429286. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and xCELLigence real-time cell analyzer were used to determine its effect on cell viability. To assess the impact of ROCK inhibitors on PTEN protein levels, PTEN expression in cell lysates was quantitatively determined in vitro using the PTEN ELISA Kit. Total protein concentration was measured using the BCA protein assay kit, p-Akt and PTEN protein level was determined by the western blot method. Showed that GSK 429286 and Thiazovivin significantly reduced U87 MG cell viability even at low concentrations. When IC values were examined, it was understood that THV (IC: 11.02 µM) showed a more potent cytotoxic effect at lower concentrations compared to GSK 429286 (IC: 89.58 µM). At 50 µM, Thiazovivin and GSK 429286 concentration caused a significant increase in PTEN activity compared to the control and Temozolomide groups, even at low concentrations. THV 50 μM significantly increased PTEN expression compared to control (p < 0.05). GSK 50 μM, THV 1 μM, and THV 10 μM groups showed significantly reduced p-Akt expression (p < 0.05). Considering the effects on healthy cells, Thiazovivin being more effective at low doses suggests it may be a more selective agent. Our findings indicate that ROCK inhibitors may have cytotoxic effects on glioblastoma cells by affecting cell death mechanisms via a PTEN-associated reduction in PI3K/AKT signaling.