The uncontrolled use of herbicides such as glyphosate (GLY) (N-phosphonomethylglycine) in agricultural production has resulted in its presence in water bodies and in negative impacts on the environment and public health. On the frame of understanding the interaction between GLY and graphite rod surfaces, this contribution relies on the study of electrochemical responses of different GLY concentrations by cyclic voltammetry under both open and closed-circuit conditions. Furthermore, the effect of the electrodes’ electrochemical pretreatment with acetic acid on the double-layer capacitance and the subsequent surface functionalization of the graphite rod materials were evaluated. The increment in GLY concentration showed a decrease in the electrochemical oxidation response associated with the adsorption of the contaminant on the surface of the graphite rod electrode and the concomitant blockage of the active sites. Electrochemical pretreatment of the electrodes with acetic acid and GLY concentration play crucial roles in electric double-layer formation due to their ability to interact with both positive and negative electrical charges. By means of optical microscope observations and Fourier Transform Infrared Spectroscopy analysis, it was possible to detect the formation of oxygenated functional groups on the electrode surfaces after the electrochemical pretreatment. Through a 23 factorial design analysis in repetition, the factors significant in the degradation of GLY were identified. The high degradation of GLY with the pretreated electrodes can be attributed to the preferential adsorption of the zwitterionic molecule at the interface, which allowed great direct oxidation of the contaminant on the anode’s surface.