The chrono-impedance technique (CIT) for real time determination of glucose concentration in a first generation glucose oxidase/carbon paste electrode was implemented. The biosensor was polarized with a signal composed of 900mV DC potential and 50mVRMS AC signal at 0.4Hz. A frequency response analyzer was used to measure the complex impedance (magnitude |Z| and phase (Φ)) of the biosensor-bulk interface. Real time measurements were performed while glucose was added to the bulk within a concentration range of 0-40mM. The cumulative impedance dose-response curves were used to construct calibration curves, both for magnitude and phase. The best fitting was obtained with a hyperbolic equation. Four biosensors were built obtaining five calibration curves for each of them. A single test measurement (unknown glucose concentration) was also obtained after each calibration procedure. Glucose concentrations were estimated with the calibration curves and also measured by colorimetry, the latter being the reference method. Besides, one-way ANOVA test evaluated repeatability. Difference between means was not statistically significant (p>0.01) for both magnitudes (|Z| and Φ). The Student's t-test assessed the differences significance, which produced in all cases p levels lower or equal than 0.44. Thus, CIT was proved to be a reliable method to measure glucose concentration in real time. Moreover, it showed high repeatability and compared well against colorimetry (r2=0.98). © 2010 Elsevier B.V.