Electrochemical sensors have great promise for point-of-care and in vivo measurement of medically relevant molecules. However, the need for calibrating individual sensors limits the practical applicability of these sensing platforms. The invention provides a novel method of operating electrochemical sensors which obviates the need to calibrate individual sensors against a sample of known concentration. The invention exploits the frequency dependence of electrochemical output signals and the dependence of output signals on the inherent electron transfer kinetics of a selected sensor design. By use of signals generated at a nonresponsive frequency, a normalizing output value is generated that accounts for sensor-to-sensor variation and which enables an accurate calculation of target concentration. The scope of the invention also includes pre-calibrated sensors that may be utilized without calibration steps.