Alcohols are widely used along with gasoline owing to their superior antiknock properties. Engine knocking depends on both fuel properties and operating conditions. The Research and Motor Octane Numbers widely used to characterize antiknock potential are calculated under specific operating conditions. In this work, a method that incorporates the effects of actual operating conditions is developed, used to determine the antiknock potential when ethanol and gasoline are used in a port fuel injected engine using simultaneous and pre-blended injection modes. Experimental results are used to build and validate an analytical model of the engine which is used to study the antiknock benefits in terms of "Effective Octane Number" (ONEff). Experiments are conducted using a newly developed injection system which utilizes separate injectors for ethanol and gasoline. This method could capture the effects of engine operating conditions in calculating antiknock benefits. Higher ethanol fractions showed better antiknock capabilities. ONEff increases from 90 to 105 units when the ethanol quantity is increased from 0 to 100% by mass, but the effect diminishes after 50%. Hence, considering antiknock benefits, the use of 50% ethanol in gasoline is recommended. ONEff of the pre-blended injection case was only 0.8 units higher, indicating the similar antiknock potential of both injection strategies. © Universiti Malaysia Pahang.