T₁AM, a derivative of thyroid hormones, and its major catabolite, TA₁, produce effects on memory acquisition in rodents. In the present study, we compared the effects of exogenous T₁AM and TA₁ on protein belonging to signal transduction pathways, assuming that TA₁ may strengthen T₁AM’s effects in brain tissue. A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG 108-15), as well as a human glioblastoma cell line (U-87 MG) were used. We first characterized the in vitro model by analyzing gene expression of proteins involved in the glutamatergic cascade and cellular uptake of T₁AM and TA₁. Then, cell viability, glucose consumption, and protein expression were assessed. Both cell lines expressed receptors implicated in glutamatergic pathway, namely Nmdar1, Glur2, and EphB2, but only U-87 MG cells expressed TAAR1. At pharmacological concentrations, T₁AM was taken up and catabolized to TA₁ and resulted in more cytotoxicity compared to TA₁. The major effect, highlighted in both cell lines, albeit on different proteins involved in the glutamatergic signaling, was an increase in phosphorylation, exerted by T₁AM but not reproduced by TA₁. These findings indicate that, in our in vitro models, T₁AM can affect proteins involved in the glutamatergic and other signaling pathways, but these effects are not strengthened by TA₁.