Flexible graphite sheets (GS) have been a promising material for developing electrochemical sensors. In this work, we showed that the electrochemical activation protocol (+1.4 V followed by -1.0 V vs. Ag|AgCl|KCl(sat.) both at 200 s) in alkaline medium (0.5 mol l-1 NaOH) enhances the electrochemical activity of GS. Before the treatment, cyclic voltammetric profile of inner-sphere [Fe(CN)6]3-/4- redox couple at 50 mV s-1 exhibited an ill-defined voltammetric profile (ΔEp = 800 mV) while an increase in the reversibility was achieved (ΔEp = 225 mV) after the treatment. Additionally, the lower charge transfer resistance (Rct) measured by Electrochemical Impedance Spectroscopy (EIS) and a higher heterogeneous electron transfer constant (k0) corroborate with the enhancement in the electrochemical performance of treated GS electrodes. The SEM images revealed an irregular pattern with number of grooves, indicating a partially surface exfoliation and AFM analysis displayed an increase in the roughness. These findings agreed with the estimation of electroactive area which increased 3-times after the treatment. As proof of concept, ketamine (KET), an anesthetic often used as drug of abuse, was determined in synthetic saliva and veterinary pharmaceutical samples using treated GS electrodes and square wave voltammetric measurements. Linear range of 5.0–100.0 µmol l-1 with a limit of detection value of 2.1 µmol l-1 was obtained for KET. Appropriate recovery values (∼105 %) for the analysis of samples were achieved.