The sterilization of microfluidic chips is a vital step of the fabrication process prior to the customer use in biomedical applications. The aim of this study was to analyze the influence of different sterilization techniques and to compare the characteristics of the material before and after sterilization of polymethylmethacrylate (PMMA) microchips. For this, supercritical carbon dioxide (SC-CO2) along with standard sterilization methods such as ultraviolet (UV), heat (autoclaving), ethylene oxide (EtO) and hydrogen peroxide (H2O2) were applied. The treated microchips were analyzed by Scanning Electron Microscopy, Differential Scanning Calorimetry, Fourier Transform Infrared Spectroscopy and Laser Scanning Microscopy in order to ascertain any changes in the chemical structure and surface morphology. The optimum sterilization parameters for SC-CO2 were elicited as 120 bar, 40 degrees C and 60 min which provided complete sterility and did not alter the main properties of the polymer along with EtO and H2O2 sterilizations unlike heat and UV treatments. However, surface roughness and microchannel profiles were negatively affected. Although complete sterility was achieved, each protocol has its own strengths and weaknesses. (C) 2015 Elsevier B.V. All rights reserved.