Phenothiazine derivatives possess biological properties very useful for cancer therapy, such as antiemetic and sedative activity as well as good blood-brain barrier permeability. Our goal was to determine if perphenazine and prochlorperazine are possessing cytotoxic activity towards U87-MG cells. It has been shown that the analyzed drugs induce concentration-dependent loss in cell viability, what correlates with their chemical structure. The calculated EC50 values for perphenazine (0.98 μM) and prochlorperazine (0.97 μM) are related to their toxic concentrations in human plasma. The obtained results suggest that perphenazine and prochlorperazine may have a potential for the development of new and effective anticancer therapies.