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Metal oxide nanoparticles have found numerous applications in different fields. In this paper, the preparation of nickel oxide nanostructures is given. The nanostructures were synthesized by using the hydrothermal method. The characterization was done with X-ray diffraction (XRD) and scanning electron microscopy (SEM). The newly synthesized nanostructures were utilized as a modifier of the working electrode, i.e., glassy carbon electrode (GCE). The modified GCE exhibited an excellent response towards methotrexate (MTX) anticancer drug. The modified GCE, as compared to bare GCE, showed an increased response towards MTX. In this study, Britton-Robinson buffer (BRB) was selected as a supporting electrolyte having pH 5. By using electrochemical impedance spectroscopy (EIS), the method was found linear in the range of 5-40 µM with a limit of detection and quantification values of 2.4 µM and 7.28 µM, respectively. The method developed by this way was successfully applied for the analysis of MTX from injection formulations. The interference studies were also carried out to check the method's selectivity.