Chip on glass (COG) bonding using Anisotropic Conductive Film (ACF) is the best process for assembling ICs on thin substrate glass with fine pitch in current practice. This paper investigates the effects of surface cleanness of the substrate, bonding pressure and contact area of COG bonding with ACF on interfacial impedance (II) via experiments. Indium tin oxide (ITO), 350±15nm in thickness, coated on 0.4mm glass is used as the substrate with φ4μm ACF particle in the experiment. Surface cleanness is measured by wetting tension, an alternative measurement of surface energy. Experiments with three levels for each parameter are designed and conducted. Reliability tests, including temperature test, humidity test, cycling test and boiling test, are also conducted to allocate best parameters in the COG bonding process. Experimental results showed that the contact area of ACF must be more than 1,500μm2 in order to provide stable II. Surface cleanness of ITO glass is suggested to be higher than 46 dyne/cm as measured by wetting tension test. Bonding pressure, ranging from 40 to 80 Mpa, does not have strong effect on II. Temperature test under -40°C /30min to 85°C/30min at 120cycle and humility test at 85°C under 85%RH for 500 hours showed that higher surface cleanness results in better bonding result as the variation of II is lower. Boiling test at 121°C under 2atm for 8 hours showed that surface cleanness should be over 46 dyne/cm, as measured by wetting tension test, to ensure stable bonding result.