Gatifloxacin is the drug of choice in the treatment of community-acquired pneumonia in many studies. However, cytotoxicity was reported at its high doses. Therefore, gatifloxacin overdose monitoring is very important. In this sense, there is a need for developing fast and cheap analytical methods for gatifloxacin quantitation in biological fluids. In the present study, a novel detection strategy involving gatifloxacin quantification in urine samples was developed. The approach has been adapted for the use of solid inner contact and rapid disposal screen-printed graphitic carbon electrodes exhibiting high sensitivity toward gatifloxacin without interference from several ions found in urine samples. The developed electrodes showed linear responses in the concentration ranges from 1 × 10–5 to 0.01 and 1 × 10–6 to 0.01 M for a solid contact glassy carbon ion selective electrode (GSC) and a screen printed electrode (GSP), respectively. The analytical applicability of the approach was demonstrated through recovery experiments of gatifloxacin trace concentrations in urine. GSP ion selective electrode (ISE) was found to have superior stability, shorter response time, higher selectivity and sensitivity and longer shelf life compared to GSC ISE. GSP ISE showed the best Nernestian slope as well as the lowest detection limit. Moreover, the inherent advantages of screen-printed electrodes technology (low sample consumption, low cost and point of care testing) make this methodology very attractive in this field. As a result, the developed ISEs can be the best choice for in-line determination of gatifloxacin in urine samples to detect overdose intake and its associated symptoms as well as for quality-control laboratories without pre-treatment or separation steps