Cell membrane permeability, one of the key biophysical properties of cells, plays an important role in bioengineering, regenerative medicine, tissue engineering, and biopreservation and biobanking. One featured and cell specific approach has been well established by the cryobiologists for determination of cell membrane permeability, i.e., osmotic shift experiments utilizing perfusion microchamber. The design of such microdevices and the corresponding experimental procedures have never been systematically discussed before, and this is the possible reason for that the measured parameters are different by different research groups. In this study, we developed a novel microfluidic perfusion chamber under the guidance of heat and mass transfer, and flow and diffusion theories. Two obvious improvements were implemented in our approach, i.e., the thermocouple was placed into the microchannel of the chamber during manufacturing to ensure that the temperature of the local extracellular solution could be accurately monitored, and being different from the previous studies, the concentration profile for the change of the extracellular solution during an osmotic shift process was carefully determined according to both experimental examination and computational finite element analysis on the fluid flow in both the capillary tube and the microchannel. Comparative experiments revealed that the commonly assumed ideal step function for the concentration changes of extracellular solution during an osmotic shift experiment inevitably introduced overlarge error when determining the cell membrane permeability by fitting the water transport equation to the experimental data, instead, the profile determined by this study effectively ensured the reliability and precision of the fitted parameters. Thus, we manufactured a novel microfluidic,perfusion chamber, and implemented the improved data processing strategy, finally, successfully developed a high-precision approach for the quantitative analysis of cell membrane permeability. Our approach has great potential for improving cryobiology research by improving the precision of experimentally determined cell membrane permeability. (C) 2015 Elsevier Ltd. All rights reserved.