Interdigitated sensors for bioimpedance analysis (BIA) are specially adapted for the characterization of low-volume (microliter scale) biological samples and the monitoring of a thin-film of biological cells for cell culture or cell settling and coagulation analysis. Impedance spectroscopy has the advantage of being a marker-free method (a combined impedance and marker is also possible), which considerably simplifies the preparation of samples. The geometry of the interdigitated sensor simultaneously represents microscopic sizes as the electrodes’ width, gap, and millimetric surface, making the sample deposition easier. The microscopic size of the electrodes induces an increase in double-layer effects, which can completely occult interesting bandwidth of the impedance measurements. This effect, therefore, must be considered early in the sensor optimization design. In this work, we propose a complete approach to optimize interdigitated sensors according to targeted applications. A complete analytical model is proposed and validated with a finite element method simulation using COMSOL Multiphysics software. The model examines the influence of all geometrical parameters, such as number of electrodes, width, gap, and substrate material. A detailed methodology is proposed to choose the best compromise between sensitivity and useful bandwidth. To validate the proposed methodology, measurements were performed on biological samples (yeast cells) using five sensors with different optimized geometries. Results demonstrated the validity of the proposed methodology and the possibility to extract all the intrinsic electrical parameters of the biological samples using both optimized sensors and our models.