Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor with a high mortality rate. Doxorubicin (DOX) and methotrexate (MTX) showed to be effective against a wide range of tumors, but its use in GBM treatment is limited in part due to the inability to cross the blood–brain barrier (BBB). Based on recent studies demonstrating that methamphetamine (METH) increases BBB permeability, we hypothesized that it could be used as a pharmacological tool to allow the entry of potential therapeutic drugs into the brain. Nevertheless, before attempting this approach it is crucial to understand the cytotoxicity of such drug combinations. Herein, we evaluated the effects of METH on cell viability, migration, chemotaxis, and cell cycle, as well as its modulator effects on DOX or MTX-induced cytotoxicity in a human U118 GBM cell line. Our results demonstrated that both chemotherapeutic drugs DOX and MTX induced a pronounced decrease in cell viability, migration, and chemotaxis, and led to a cell cycle arrest at G₂ and S phases, respectively. Additionally, METH (1 μM) neither interfered with U-118 cell viability, migration, or cell cycle nor modified DOX- or MTX-induced cytotoxicity. Noteworthy, METH by itself impaired cell chemotaxis with a similar effect to that induced by DOX or MTX alone. Overall, we can conclude that both DOX and MTX are highly cytotoxic against GBM cells and that METH, at a concentration previously shown to increase endothelial cell permeability without leading to cell death, does not interfere with the cytotoxicity of both chemotherapeutic drugs.