AbstractTrimethoprim (TMP) is a bacteriostatic antibiotic mainly used in the prophylaxis and treatment of urinary tract infections. It belongs to the class of chemotherapeutic agents known as dihydrofolate reductase inhibitors. Its use is associated with idiosyncratic reactions, including liver toxicity and agranulocytosis. In order to determine TMP electrochemically, a metalloporphyrin modified glassy carbon electrode was prepared by coating [5,10,15,20‐ tetrakis(4‐methoxyphenyl) porphyrinato]Mn (III)chloride (TMOPPMn(III)Cl) solution on the surface of the electrode. The electrochemical behaviour of TMP in Phosphate buffer solution (PBS) on TMOPPMn(III)Cl modified glassy carbon electrode (TMOPPMn(III)Cl/GCE) was explored using differential pulse voltammetry (DPV). The voltammograms showed enhanced oxidation response at the TMOPPMn (III)Cl/GCE with respect to the bare GCE for TMP, attributable to the electrocatalytic activity of TMOPPMn(III)Cl. Electrochemical parameters of the oxidation of TMP on the modified electrode were analyzed. The electro‐oxidation of TMP was found to be irreversible, pH dependent and adsorption controlled on the modified electrode. It is found that the oxidation peak current is proportional to the concentration of TMP over the range 6 × 10−8 − 1 × 10−6 M with a very low detection limit of 3 × 10−9 M at 2 min open circuit accumulation. The repeatability expressed as relative standard deviation (RSD) for n = 9 was 3.2% and the operational stability was found to be 20 days. Another striking feature is that equimolar concentration of sulfamethoxazole did not interfere in the determination of TMP. Applicability to assay the drug in urine and tablet samples has also been studied. Copyright © 2010 John Wiley & Sons, Ltd.