We report on a thin layer coulometric detection method based on an ion-selective membrane in view of a quantitative determination of nitrite in undiluted human urine and tap water. A cobalt(II) tert-butyl salophen compound, previously characterized by our group in ion-selective membrane electrodes, is used here as ionophore. The four orders of magnitude discrimination of chloride permits the detection of nitrite in samples that contain a large excess of chloride. The approach developed here allows one to determine nitrite within a concentration range of 20–100 µM for a 120 s current integration time. A modification of the reported electrochemical protocol to reduce the capacitive contribution diminishes the intercept of the calibration to −8.6 µC. This is especially important in view of reaching calibration free systems. Initial in-house validation exhibits a limit of detection of 10 µM in undiluted urine and in tap water. These values correspond to the regulation limits established in European Union and are adequate for the determination of nitrite for medical applications.