high performance liquid chromatography (HPLC) with isocratic ion-pair-reversed-phase separation and UV detection at 230 nm is proposed for the determination of creatinine in urine. The analysis is achieved on a C18-Symmetry column (4 μm, 150mm × 3.9mm ID) using a mixture of 10 mM phosphate buffer pH=4, containing 3mM 1-octane-sulfonic acid, sodium salt and methanol (97:3). A flow-rate of 0.5mL/min and a column temperature of 30°C were employed. We validated the method for linearity, precision, and accuracy and the results were found to be excellent. The procedure proposed here is an alternative to less specific colorimetric methods.