The present study aimed to investigate the effect of N-Ethylmaleimide (NEM) on the evaluation of disulfide formation in the oxi  dized myofibrillar proteins during the sample preparation of the non-reducing SDS-PAGE procedure. For this purpose, extracted myofibrillar proteins were oxidized firstly via a Fenton oxidation reaction, and non-oxidized proteins were used as a control. Before running SDS-PAGE, in the sample preparation, these oxidized and non-oxidized proteins were prepared according to the three dif  ferent sample preparation methods with or without the presence of N-Ethylmaleimide or β-mercaptoethanol. Results showed that oxidized proteins treated with NEM regardless of sample preparation methods presented attenuated bands of myosin heavy chain monomer in the non-reducing SDS-PAGE gels, suggesting that the disulfide bonds formed as a result of protein oxidation could be preserved by NEM during sample preparation. Meanwhile, a possible mechanism for the effect of NEM was proposed.