- We present the first in-depth system integration study of in-plane hydrodynamic focusing in a microfluidic impedance cytometry lab-on-a-chip. The method relies on constricting the detection volume with non-conductive sheath flows and characterizing particles or cells based on changes in impedance. This approach represents an avenue of overcoming current limitations in sensitivity with translating cytometers to the point of care for rapid, low-cost blood analysis. While examples of integrated devices are present in the literature, no systematic study of the interplay between hydrodynamics and electrodynamics has been carried out as of yet. We develop analytical and numerical models to describe the impedimetric response of the sensor as a function of cellular characteristics, physical flow properties, and device geometry. We fabricate a working prototype lab-on-a-chip for experimental validation using latex particles. We find that ionic diffusion can be a critical limiting factor even at high Péclet number. Moreover, we explore geometric variations, revealing that the ionic diffusion-related distance between the center of the hydrodynamic focusing junction and the impedance measurement electrodes plays a dominant role. With our device, we demonstrate over fivefold enhancement in impedance signals and population separation with in-plane hydrodynamic focusing. It is only through such in-depth system studies, in both models and experiments, that optimal utilization of microsystem capabilities becomes possible.