Background: The plasma membrane is a highly fluid and dynamic structure. It is a lipid bilayer mainly composed of phospholipids. It has many different roles that affect cell function. One of the fundamental biochemical differences between tumor cells and normal cells is the composition of the cell membrane’s lipid content. Cancer research has focused on the chemotherapeutic effect inside the cell without taking into consideration the effect of these drugs on the cell’s plasma membrane. Methotrexate, a folic acid analogue used in cancer chemotherapy, is a potent inhibitor of dihydrofolate reductase (DHFR), a key enzyme for the biosynthesis of purine and thymidine. It is used as a chemotherapeutic agent for human malignancies such as acute lymphoblastic leukemia, malignant lymphoma, osteosarcoma and breast cancer. Aim: The aim of this study is to examine the effect of methotrexate on the phosphatidylserine and phosphatidylethanolamine composition of the plasma membrane of MCF-7 breast cancer cells and to evaluate its effect on the invasiveness of these cells in an in vitro system. Materials and Methods: The effect of methotrexate on the viability of MCF-7 breast cancer cells was evaluated using MTT assay using concentrations ranging from 0.1μM to 50μM. The cells were treated with methotrexate at IC50. The plasma membrane of both control and treated cells was isolated and their proteins were quantified. Inorganic phosphate was determined for the lipids extracted from MCF-7 control and methotrexate treated cell homogenate as well as their isolated plasma membrane. The extracted lipids were separated using high performance liquid chromatography. The effect of methotrexate on the cells’ invasiveness was assessed using time lapse assay. Results: • Methothrexate induced 50% cell death at 5 μM as shown by MTT. • The presence of 5’-nucleotidase and the absence of glucose-6-phosphatase and succinate dehydrogenase ensured the plasma membrane purity from any cellular organelles. • There was no significant change in the total protein content between treated and control cells, while it doubled in the plasma membrane of the treated cells compared to that of the control cells. • The amount of inorganic phosphate in total homogenate and plasma membranes decreased upon treatment with methotrexate. • The inorganic phosphate of the extracted plasma membrane lipids showed no significant change after treatment with methotrexate. • The chromatograms of extracted lipids showed no significant change between control and treated cells except for the lipid eluted at a retention time of 4.36 min. • The motility of MCF-7 breast cancer cells treated with 5μM methotrexate decreased 17.4% compared to the control.